Posts Tagged ‘biosimilars’

RAbD Biotech Presents at 1st Pitch Life Sciences-Philadelphia-September 16, 2014

Reporter: Stephen J. Williams, PhD

UPDATE 12/09/2022

The following is background material of RAbD technology, bispecific antibodies:

Current landscape and future directions of bispecific antibodies in cancer immunotherapy

Jing Wei1†Yueyao Yang2†Gang Wang2 and Ming Liu1*
  • 1Gastric Cancer Center/Cancer Center, West China Hospital, Sichuan University, Chengdu, China
  • 2National Engineering Research Center for Biomaterials, Sichuan University, Chengdu, China

Recent advances in cancer immunotherapy using monoclonal antibodies have dramatically revolutionized the therapeutic strategy against advanced malignancies, inspiring the exploration of various types of therapeutic antibodies. Bispecific antibodies (BsAbs) are recombinant molecules containing two different antigens or epitopes identifying binding domains. Bispecific antibody-based tumor immunotherapy has gained broad potential in preclinical and clinical investigations in a variety of tumor types following regulatory approval of newly developed technologies involving bispecific and multispecific antibodies. Meanwhile, a series of challenges such as antibody immunogenicity, tumor heterogeneity, low response rate, treatment resistance, and systemic adverse effects hinder the application of BsAbs. In this review, we provide insights into the various architecture of BsAbs, focus on BsAbs’ alternative different mechanisms of action and clinical progression, and discuss relevant approaches to overcome existing challenges in BsAbs clinical application.

1 Introduction

Immunotherapy breakthroughs in cancer treatment with synthetic multifunctional biotherapeutics have fueled cancer immunotherapy and the exploration of antibody alternative modes of action (12). Conventional targeted or immunotherapeutic drugs can only be used to inhibit one or one class of targets, thus giving birth to some unique combination drug regimens. Currently, Constant engineering technical breakthroughs in antibody development have aided in producing many BsAb designs (3) (Figures 1C, D). Bispecific antibodies, constructed via quadroma, chemical conjugation, and genetic recombination (4), exert effector functions beyond natural antibodies through redirecting cells or modulating different pathways, providing numerous possibilities for therapeutic application and contributing to improving treatment responses in refractory tumor patients. Although more than a hundred BsAbs are currently under clinical evaluation in cancer treatment, most are still in the early stages (4), and only four BsAbs have been approved by FDA (Table 1). These include Catumaxomab (Fresenius/Trion’s Removab®) which was withdrawn from the market in 2017, Blinatumomab (Amgen’s Blincyto®), Amivantamab-vmjw (Janssen’s Rybrevant®), and Tebentafusp-tebn (Immunocore’s Kimmtrak®) (511).

Figure 1
www.frontiersin.orgFIGURE 1 CD3+ bispecific T-cell engaging antibodies exert function in Hematological Malignancies and recruit immune cells into the solid tumor microenvironment for cancer immunotherapy. The schematic depicts the mechanism of action of BsAbs in solid tumors (A) and Hematological Malignancies (B). besides, there shows partial fragments of antibody as well as the derivatives formats constructed from them in diagram (C) and various architecture of BsAbs in diagram (D) mentioned in this review.

Table 1
www.frontiersin.orgTABLE 1 Bispecific Antibody Approved by the FDA.

Furthermore, multiple studies investigate the mechanisms of action by which BsAbs detect various tumor targets such as angiogenesis, proliferation, invasion, and immune modulation. However, potential immunotherapy side effects must be considered, whereas toxicity in normal tissues and systemic immune responses limit the use of BsAbs (1215). We concentrate on the advances in BsAbs design, mechanisms of action, and clinical trial development in this review (Table 2). We also talk about difficulties and potential solutions for enhancing drug delivery.

Table 2
www.frontiersin.orgTABLE 2 Bispecific Antibody Clinical Trials Ongoing.

2 BsAb construct formats

In natural bivalent antibodies, the two antigen binding sites are identical and consist of variable regions of the heavy chain and light chain. Bispecific antibodies (BsAbs) are dual-specificity molecules binding two different epitopes simultaneously, the concept of which has been first described decades ago by Nisonoff et al. (1). Since there are no naturally occurring bispecific antibodies, BsAbs were initially developed by chemically coupling two monoclonal antibody fragments or creating quadroma cell lines combined with two homologous hybridomas. The field of recombinant bispecific antibodies for diagnostic and therapeutic purposes has been transformed by the quickly developing engineering technologies and pharmaceutical industry, leading to a variety of BsAbs with varying size, half-life, valency, flexibility, and permeability (2). Recombinant DNA technology is now the most used technique for producing bispecific antibodies.

IgG-like antibody types (containing an Fc unit) and non-IgG-like (without an Fc unit) antibody formats are the two broad categories into which BsAbs can be generally categorized (3). The intention of this classification mechanism well emphasizes the existence of the Fc domain, which not only facilitates the functionality mentioned above but contributes to the solubility, stability, and purification of the BsAbs (4). Additionally, this region can be genetically altered to abolish antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) while retaining the potential for a lengthy half-life (5). Although non-IgG-like antibody formats exert therapeutic activities depending on antigen-recognition domains, smaller size enables them to enhance tissue penetration while rapid renal clearance results in a relatively short plasma half-life.

Heavy and light chain mispairing poses serious problems for bispecific antibodies made in IgG formats from two distinct polypeptides, leading to ineffective antibodies or unwanted homodimers. In the interim, mitigation measures have been taken for such issues. For example, the “knobs-into-hole” approach has been developed to mutate the corresponding amino acid size of the third constant domain of the antibody for correctly pairing the heavy chain (6). In contrast, “the light chain mispairing” problems can be well circumvented by the “CrossMab” strategy by swapping the CL (light chain constant region) domain of the light chain with the corresponding CH1 domain of the heavy chain to construct a correct light chain association (712). Other established and mature techniques to prevent light chain mispairing include Eli Lilly’s “Orthogonal Fab interface” by introducing amino acid mutations to the light chain and the tcBsIgG (tethered-variable CL bispecific IgG) platform developed by the Genentech, which focuses on linking the VL (light chain variable region) to VH (heavy chain variable region) via the G4S linker (89). In addition, the asymmetric heavy chains also form the basis for constructing multi-specific antibodies, such as trispecific, trivalent, or tetravalent antibodies, with greater targeting specificities for cancer therapy.

The production of non-IgG-like BsAbs without an Fc unit can be accomplished using Fab fragments or by joining the variable light-heavy domains of two antibodies. These antibodies can be broadly categorized into scFv (single-chain variable fragment)-based bsAbs, Nanobodies, Dock-and-lock (DNL) method-building antibodies, and other bispecific/multi-specific molecules (10). The specificity and ability to bind to antigens of full-length antibodies are maintained by scFv, composed of the VL and VH domains connected by a flexible peptide. Additionally, the linker length significantly impacts how scFv molecules associate, resulting in various polymerizations such as dimers, trimers, or tetramers. The Tandem scFvs constructed by the Bispecific T-cell Engager (BiTE®) antibody platform link two scFvs with a repeat glycine-serine short motifs (11), which enables antigen recognizing sites to rotate flexibly.

In contrast, the linking method of the Diabody format is slightly different from Tandem scFvs (13). Heterodimerization of those fragments is induced by crossing the VH and VL domains of the two scFvs by two shorter polypeptide chains. Even if the two peptides linker facilitates the molecular stability to a certain extent, the intramolecular association between the VH and VL domains from the same scFv is hampered (14).

Further research on crystal structures suggests that the Fv contact’s instability impacts overall flexibility, and the diabodies are too rigid to crystallize. As a result, “Dual-affinity retargeting molecules”—an inter-chain disulfide bond—have been created to strengthen the structural stability of the diabodies (DARTs) (1516). However, the small size of this format makes the DARTs molecule rapidly eliminate from the serum, and this issue has been well solved by the strategy of Fc fragment fusion to the part of the DARTs, promoting FcRn-mediated recycling at the same time. The emergence of this constructive format of bispecific antibodies called “DART-Fc” was designed by MacroGenics. Besides, the polymerization of two diabodies connected by two polypeptide chains forms the “Tandem diabodies” (TandAbs), the tetravalent derivatives possessing two antigen-recognizing moieties for each antigen.

In addition to being synthesized from the constituents of various antibodies, BsAbs can be fused to other protein domains to improve their functions for future adaptive therapeutic uses. For example, the scFv-based BsAbs have relatively great tissue permeability and decreased immunogenicity for the lack of Fc unit. However, short half-lives caused by relatively low molecular mass will affect the serum circulating level, which induces the Increased administration and doses of therapeutic agents, thus limiting the clinical promoting application. The format “scFv-HSA-scFv”, fusing two scFvs to the albumin, and conjugation of Polyethylene glycol PEGylation are available application strategies for circulating half-life extension (17). Furthermore, the Dock-and-lock (DNL) method, creating multivalent and multifunctional antibody derivatives such as trivalent bispecific antibodies by heterodimerizing protein domains fused with Fab domains or integral antibodies, is another available antibody platform enabling more promising antibody construction with retained bioactivity (18) (Figure 1C).

3 BsAbs redirecting immune effector cells and reactivating anti-tumor immunity

3.1 Bispecific T-Cell Engagers (BiTE) recruiting adaptive effector cells for tumor redirection

Adaptive immunity is essential for monitoring and suppressing tumorigenesis and cancerous progression. T-cells are the focal point of many immunotherapies and have a potent tumor-killing effect as a crucial part of adaptive immunity. BiTE is a potential immunological drug that directs T lymphocytes against tumor cells to treat a variety of malignancies. The ideal BiTE molecular design needs to consider the following factors: firstly, a suitable CD3 binding arm should be selected; secondly, the affinity between TAA binding sites and anti-CD3 units should differ by at least 10-fold to achieve sequential binding to tumor cells and immune cells, thus reducing the risk of severe cytokine release syndrome (CRS); finally, an appropriate physical distance between the two antibody binding units can also play a role in improving the efficacy and reducing the risk of CRS.

The high affinity of CD3 binding will allow BiTE to occupy CD3 antigen and activate T cells continuously, eventually leading to T cell depletion. However, suitable affinity will bind to and dissociate from the CD3 antigen of distinct T cells and the repeated binding to T cells will cause a “waterfall effect” to cluster T cells, ultimately resulting in many T cells activating to attain the greatest tumor-killing effect. In addition, high CD3 binding affinity will make BiTE more concentrated in the spleen, thus making it difficult to reach tumor tissues, especially solid tumors. Physically bridging T cells with tumor cells by BiTEs enables catalyzing the formation of an optimal immunologic synapse, which’s important for T cells activation and robust cytotoxicity to target tumor cells, ultimately leading to apoptosis via membrane disruption mediated by perforin releasing. In addition, multiple cytokine secretion from T cells activated by BiTEs, like IL-2, IFN-γ, and TNF-α, enhance their effectiveness in the anti-tumor function (Figures 1A, B). Strategies that harness the potential of T cells to identify and kill cancer cells in a targeted manner have ushered in a new era of cancer therapy and led to the development of a wide range of immunotherapy devices.

3.1.1 BiTEs in hematologic malignancies

Due to the distinct traits of hematopoietic malignancies, immunotherapy for leukemia and lymphoma has assumed a leadership role and made significant advancements. Given the unique property of the hematological system, malignancy cells constantly interact with immune cells, making it easier for BiTE to exert anti-tumor actions (19). Among various forms of immunotherapy, engaging T cells in hematological malignancies mediated by bispecific antibodies (BsAbs) has been demonstrated as an attractive strategy for providing alternative treatment options for recurrent and/or refractory hematological malignancies patients (20). Bispecific T-cell engagers (BiTEs), consisting of two binding sites simultaneously for a selective tumor antigen and CD3 molecule expressed on host T cells, has been emerged as the most promising BsAb form (Figure 1B). A diverse variety of BiTEs have emerged for cancer immunotherapy, and the specific targets are mainly CD19, CD20, CD123, CD33, CD38, and B-cell maturation antigen (BCMA) in hematological malignancies. Ideal target antigens must satisfy the criteria uniquely expressed on malignant cells to avoid on-target/off-cancer toxicity and reduce the possibility of antigen-loss variants. Even though few target antigens satisfy the above demands simultaneously (2122), various types of BiTE with different antigen-recognition domains have been under exploration for hematological malignancies.

Blinatumomab (MT103), the first BiTE tested in clinical trials specifically designed to target T cells based on the recognition of CD3ϵ to CD19 expressing B cell hematologic malignancies, can induce immune-mediated B-cell lymphoblasts lysis led by cytotoxic T cells. CD19 is a transmembrane molecule relatively specific to B cells persisting throughout B-cell differentiation and existed on the surface of most B cell hematologic malignancies, which is a superior target for cancer immunotherapy achieving exceptional curative effect with R/R B cell ALL (Acute Lymphocytic Leukemia) patients. And the body’s function will not be seriously affected while the missing of normal B cells or bone marrow cells can be continuously replenished by hematopoietic stem cells.

A 7.1-month overall survival and a CR/CRh of 36%, including partial patients with a T315I mutation, were achieved in an open-label phase II study evaluating the efficacy and tolerability of Blinatumomab in Ph-positive (Ph+) B-precursor ALL in the setting of relapsing or refractory to TKI-based therapy. This study findings helped the FDA expand its approval for Ph+ ALL indication in July 2017 (23). MRD is the most powerful prognosticator of relapse in ALL, and MRD-negative status has become increasingly clear that significantly associated with better event-free survival (2425). series of clinical trials have demonstrated the potency and efficacy of Blinatumomab in eradicating persistent or relapsed MRD in B-ALL patients with an increased MRD response rate (2630). Based on these encouraging clinical findings, Blinatumomab acquired accelerated approval by the FDA to expand clinical indications to patients with MRD-positive ALL in 2018 (30). Blinatumomab has also been studied in phase 1/2 dose-escalation experiments for R/R Non-Hodgkin lymphoma (NHL), including diffuse large B-cell lymphoma (DLBCL), reaching an overall response rate of more than 40% (3132).

Although the clinical benefits for B-ALL patients from Blinatumomab is obvious, there are still problems with severe neurological events (encephalopathy, aphasia, and seizures) and cytokine release syndrome. In most studies, about 10% of patients reported ≥ grade 3 CRS and/or neurological complications. Dexamethasone or treatment interruption can alleviate these unfavorable side effects. In spite of this, about 10% of patients discontinued with Blinatumomab application because of treatment-related toxicity. Lunsumio (Mosunetuzumab) was recently granted conditional marketing authorization by the European Commission, a CD20 × CD3 T-cell binding bispecific antibody, for treating adult patients with relapsed or refractory follicular lymphoma (FL) who have received at least two prior systemic treatments. Roche is also working on Glofitamab, a CD20 × CD3 bispecific antibody with a different structure than Mosunetuzumab. Mosunetuzumab is similar to a natural human antibody but contains two different Fab regions, one of which targets CD20 and the other targets CD3. Glofitamab has a novel “2:1” structural pattern with two Fab regions targeting CD20 and one Fab region targeting CD3. This novel structural design allows higher binding to CD20 at the B cell surface.

The classification of hematological tumors is complex and varied. Acute myeloid leukemia (AML) is a genetically diverse disease defined by leukemic cell clonal proliferation. BiTEs are an effective treatment for AML because AML cells are especially vulnerable to the cytotoxic effects of functioning immune cells. CD33 expression is limited in non-hematopoietic tissues but is highly expressed in AML cells. The differential expression of CD33 on the surface of malignant AML cells makes it an ideal target for immunotherapy. AMG 330, the first CD33 × CD3 BiTE applied for acute myeloid leukemia (AML) patients, has shown promising cytolytic activity against AML cells in preclinical studies even at low CD33 antigen densities on target cells, making it a candidate for targeting a broad range of CD33+ leukemias (3335). AMG330, similar to Blinatumomab, requires a 2-4 week cycle of continuous intravenous (IV) infusion. AMG330 was found to upregulate PD-L1 on primary ALL cells in vitro. As a result, when paired with PD-1/PD-L1 blocking therapy, AMG 330-mediated tumor cell lysis was dramatically increased (3637).

A bifunctional PD-1 × CD3 × CD33 immune checkpoint inhibitory T-cell engaging (CiTE) antibody simultaneously targeting PD-1, CD3 and CD33 has shown high therapeutic effect with complete AML (Acute Myelocytic Leukemia) eradication in preclinical experiments (38). Many clinical trials are ongoing with combination therapy of bispecific T cell-engaging antibodies and PD-1/PD-L1 axis inhibitors. The administration period depends largely on the structure of the antibody. The design of bispecific antibody-like Blinatumomab needs to consider the half-life. AMG673 will have an increased half-life of about 21 days in humans after fusing the binding domain of CD33 and CD3 to the N-terminal end of the IgG Fc region. In this way, the cycle time for intravenous infusion can be reduced. However, more attention needs to be paid to adverse effects. The drug AMV564 has a higher affinity for both antigens and possesses a tetravalent anti-CD33 × anti-CD3 tandem diabody (TandAb) structure with two CD3 binding sites and two CD33 binding sites. Whether T cells are overactivated is a concern for AMV564. Compared to AMG330, AMV564 is administered by continuous intravenous infusion at 14-day intervals. Preclinical studies in vitro and in vivo have demonstrated the ability of AMV564 to induce effective cytotoxicity to CD33+ AML cell lines in a dose-dependent manner.

CRS is the main toxic reaction in patients treated with CD33-targeted bispecific antibodies. Still, differences in the frequency and severity of CRS may depend on the leukemic load, the effector target ratio at baseline, the specific bispecific antibody structure, and its affinity for CD3.

More bispecific antibody for AML is undergoing clinical trials, such as CD123 × CD3 DuoBody (NCT02715011) and CD123 × CD3 DART (NCT02152956), and preclinical evaluation for adult patients, such as targeting CLL-1 and CD47. Target selection and efficacy assessment must be more cautious when treating AML in pediatric patients. More than 30% of AML pediatric patients have a highly tumor-specific target called MSLN. Recently, BsAbs targeting the MSLN and CD3 proximal area epitopes have increased lifetimes by increasing T cell activation and decreasing the tumor’s bone marrow AML cell load in MSLN-positive mice (39).

3.1.2 BiTEs in solid malignancies

Bispecific T cell engagers (BiTEs) have revolutionized success in hematological malignancies treatment and revitalized the field of solid tumor immunotherapy with promising outcomes from preclinical and clinical trials. Even if the checkpoint inhibitors hold the majority of approvals in recent years in various solid tumor types, the T cell redirection and recruitment approaches are extremely promising. Contrarily, the solid tumor microenvironment has incredibly complex features that affect the infiltration, activity, and persistence of immune effector cells vital to anti-tumor immunotherapy (311) (Figure 1A).

Catumaxomab (Removab) was the first bispecific T cell engagers (TCE) approved by the European medicines agency (EMA) for malignant ascites clinical intraperitoneal treatment in 2009 (4041). It is the intact trifunctional mouse/rat chimeric bispecific IgG antibody, with one arm from mouse IgG2a half-antibody identifying epithelial cell adhesion molecule (anti-EpCAM) on tumoral cells and another arm from rat IgG2b targeting CD3 subunit (anti-CD3) on T-cells. Additionally, the functional Fc fragment binds to different immune accessory cells with Fcγ receptors (FcγR), such as natural killer (NK) cells, dendritic cells (DC), monocytes, and macrophages resulting in T-cell-mediated lysis, ADCC, and accessory cells mediating phagocytosis (42). It employs humoral immunity, on the one hand, activates cellular immunity on the other, and delivers co-activation signals via attachable immune cells to eliminate truculent tumor cells, as well as allowing the body’s immune system to generate a specific immune memory, which acts as a cancer vaccine and inhibits tumor metastasis and recurrence. Catumaxomab’s effectiveness has been proven in key phase I/III research and other phase I/II studies (40).

However, intravenous applications of Catumaxomab were connected with severe adverse events like cytokine release syndrome (CRS) and dose-dependent liver toxicity (43), attributing to the off-target activity of other immune cells with FcγRs expressing, and it was withdrawn in 2017 from the market for some commercial reasons (44). The efficacy, safety, and tolerability of Catumaxomab are currently being studied in clinical trials for various indications involving patients with non-muscle invasive bladder cancer (NMIBC) and advanced or recurrent gastric carcinoma with peritoneal metastasis (NCT04819399; NCT04222114) (45).

Driven by the clinical success of Blinatumomab, various T cell-engaging BsAbs targeting solid tumors have been explored and evaluated in preclinical mouse xenograft tumor models and clinical trials. Various antigen being investigated for CD3 TCE bsAbs such as CEA, HER2, prostate-specific membrane antigen (PSMA), GlycoproteinA33 (gpA33), and Glypican 3 (GPC3), etc. (4649). On-target off-tumor toxicity, a restricted number of effector cells in the tumor microenvironment (TME), and decreased T cell activation in tumors are all problems with CD3 bispecific antibodies in solid tumors. Anti-bispecific antibody designs and techniques for numerous challenges are also available. As a result, several novel formats of TCEs and prodrugs have been investigated for improved efficacy, such as a TCE with a monovalent CD3 binding region and a multivalent TAA binding region, which has been shown to effectively transform a poorly infiltrated tumor microenvironment (TME) into a highly inflamed TME with increased infiltration frequency of activated T cells. Besides, these new multivalent TAA binding regions enable avoiding on-target/off-tumor toxicities after being altered into low-affinity TAA binding domains since most TAAs are also generally expressed at low levels in normal tissue cells (5051).

Dionysos Slaga and colleagues have demonstrated a proof of concept. They have generated an anti-HER2/CD3 TCE BsAbs which can target HER2-overexpressing tumor tissue cells with selectivity and high potency while very low binding to normal tissue cells with low amounts of HER2 expressing, thus circumvents the risk of adverse effects to a certain extent (52). Current prodrugs utilize the characteristics of tumor microenvironment such as lower pH, oxygen levels, and proteolytic enzyme levels, allowing for tumor-specific activation of BsAb that are inactive in the circulation or normal tissue thus avoiding attack normal cells. Furthermore, the most fundamental strategy to avoid on-target/off-tumor toxicity is to choose targets only expressed in solid tumors. Immune-mobilizing monoclonal TCRs against cancer (ImmTAC) is a remarkable TCE bsAb format that targets MHC-presented intracellular neoantigen peptides on the surface of tumoral cells (53). This engineered antibody format consisted of an anti-CD3 scFv and TCR peptides with enhanced affinity, enabling to recruit and selectively activate a majority of polyclonal effector T cells to infiltrate to reverse the “cold” TME into an inflammatory TME and lysis cancer cells with low surface oncoprotein epitope densities in the context of HLA-A*0201 subsequently, which demonstrates significant anti-tumor efficacy (54). Tebentafusp (IMCgp100), an ImmTAC molecule, for example, targets melanocyte differentiation antigen polypeptide glycoprotein100 (gp100), showing clinical activity in Metastatic Uveal Melanoma with low tumor mutational burden. Tebentafusp outperformed a single-agent treatment of ipilimumab, pembrolizumab, or dacarbazine in an open-label phase 3 trial for extending overall survival in newly diagnosed patients with metastatic uveal melanoma (NCT03070392) (55).

Whether changing the valence of antibodies, bispecific antibodies in the form of prodrugs, or finding new targets can only solve the problem of on-target off-tumor, T cell infiltration and activity in solid tumors can mostly only be changed by the mode of administration or combination therapy. TAA-based targeting may underestimate the use of CD3 bispecific antibodies in solid tumors. A bispecific antibody against both PD-L1 and CD3 successfully connected T cells to PD-L1-expressing tumor cells, improved T cell cytotoxicity against multiple NSCLC-derived cell lines by releasing granzyme B and cytokines, and decreased tumor growth in mice (56). However, a more potent mechanism of action may exist for the PD-L1 × CD3 bispecific antibody, which was found to target dendritic cells rather than tumor cells in multiple homozygous tumor mouse models. Bispecific antibodies redirecting T cells to APCs by enhancing B7/CD28 co-stimulation to activate T cells may represent a general means of T-cell rejuvenation for durable cancer immunotherapy. PD-L1 × CD3 treatment is undoubtedly dual-acting by simultaneously blocking negative signaling (PD-L1) and engaging positive signaling (CD3). More targeting approaches are now conceivable with the identification of immune checkpoints, and more mechanisms of action are being examined (57).

Any single technique for treating solid tumors will either limit tumor growth or temporarily remove the tumor. A combination of multiple techniques is required to achieve the optimal treatment result. More target combinations and antibody screening modalities are being developed for solid tumors. Using Patient-derived organoids (PDOs), bispecific antibodies can be screened on a large scale, and their efficacy can be evaluated more reliably than in cell experiments.

3.2 BsAbs recruiting Natural Killer (NK) cells for tumor redirection

NK cells, derived from multipotent hematopoietic stem cells, were identified in 1975 and have been considered the first line of defense against tumor cells with the robust anti-tumor ability (5859). With MHC-independent cytotoxicity, cytokine synthesis, and immunological memory, NK cells have a unique anti-tumor function, making them crucial participants in the innate and adaptive immune response system. These cells are conventionally divided into two subtypes, CD56dim CD16+ NK, and CD56bright CD16 NK cells. The former possesses powerful cytotoxicity and constitutes most of the peripheral blood and spleen subpopulation. At the same time, the latter is mainly equipped with immunomodulatory characteristics and constitutes a major subtype in lymph node tissues with weak cytotoxicity and maturity.

Diverse inhibitory and activating receptors are expressed on the surface of NK cells, determining the outcome of NK-cell activation by mediating the balance between those signals, which is pivotal for distinguishing and eliminating aberrant from normal cells through cytotoxic granules secretion based on TRAIL receptors and FAS ligand (FasL) expression as well as the release of other cytokines, growth factors, and chemokines. Inhibitory receptors on the surface of NK cells can recognize and bind to MHC Class I (MHC-I) molecules to alleviate autoimmune reactions. In contrast, the down-regulated expression of the MHC-I molecule can induce NK cell-mediated killing under cellular stress conditions, known as “missing self-recognition”. During tumorigenesis, the expression of MHC-I molecules is generally lost or in a defective condition for escaping from immune surveillance. Still, the unique characteristic of NK cells plays a necessary role in bypassing downregulated presentation of tumor neoantigens and effectively eliminating early aberrant cells (58).

A high frequency of NK-cell infiltration is usually connected with a better prognosis. However, Clara Degos and colleagues found an impoverished NK cell infiltration in the tumor microenvironment. IFN-γ secretion and cytotoxicity of Tumor-infiltrated NK cells are impaired, with significantly attenuated tumor-killing ability (60). Bispecific killer cell engager (BiKE) is a promising strategy to engage NK cells to tumor cells; Fc receptor (FcγRIII, CD16A)-mediated recruitment as a function of bsAb can be achieved by binding of CD16 on the surface of NK cells to the Fc region of bsAb, or by one end of a bi-specific antibody targeting CD16A (CD16A antibody). By inducing ADCC, the activating NK cell receptor CD16A (FcγRIIIA), which is mostly expressed on mature NK cells, might facilitate the destruction of tumor cells (61).

Both the composition and form of the BiKE affect the effectiveness of ADCC induction. The design of BiKE for Fc-mediated NK cell recruitment faces the challenge that the chosen form needs to ensure the effective binding of the Fc structural domain to CD16. Same as the Fc-mediated ADCC antibodies, the ADCC efficiency of BiKE, which recruits NK cells through partial regional CD16 antibody, is also similarly dependent on the choice of antibody’s form. ADCC-induced binding of the tumor-associated antigen (TAA) CD30 and CD16A is superior to monovalent CD30/CD16A binding in the dual CD16A-bound TandAb (tandem diabody) form than in the diabody form (62).

The AFM13 (ROCK®), a tetravalent bispecific anti-CD30 × anti-CD16A TandAb targeting CD30+ malignancies like Hodgkin lymphoma, has shown efficacy and cytotoxicity in an early clinical trial (NCT01221571) (63). Besides, in a phase 1b study aiming to evaluate the curative effect of AFM13 in combination with pembrolizumab to investigate further a rational treatment modality in patients with relapsed/refractory Hodgkin lymphoma (R/R HL), the objective response rate reached 88% at the highest treatment dose. The overall response rate is 83% for recipients with an acceptable safety profile and tolerability (64). Furthermore, AFM13 has reached a phase II clinical study to improve therapeutic efficacy by optimizing the dosing schedule (63). The AFM24, a different CD16A-based IgG1-scFv fusion BsAb that targets EGFR-expressing tumor cells with varying EGFR expression levels and KRAS/BRAF mutational status, has also demonstrated a strong potential for therapeutic application investigation and is now being studied in clinical trials (NCT04259450) (65).

Apart from targeting CD16A, a small number of studies have targeted activation receptors on NK cells, such as NKG2D, NKp30, and NKp46. A study constructed a homodimeric recombinant antibody combining two NKG2D-binding and two ErbB2 (HER2)-specific single-chain fragment variable (scFv) domains, linked by an IgG4 Fc region in a single tetravalent molecule, known as NKAB-ErbB2 (66).

The NKAB-ErbB2 increased lysis of ErbB2-positive breast carcinoma cells by peripheral blood-derived NK cells endogenously expressing NKG2D. NKG2D is unlike the other targets because mAb-mediated cross-linking does not result in cytokine release. In contrast, stimulation with soluble recombinant NKG2D ligands (MICA, ULBP-1, or ULBP-2) induces the expression of IFN-γ, GM-CSF and MIP-1β. A novel dual-targeting antibody composed of antibody cG7 and MICA was named cG7-MICA. The cG7 part is a natural antibody targeting CD24, and MICA is attached to the antibody behind CH3 via a G4S linker. When cG7-MICA coupled to CD24 on tumor cells, inducing NK cell-mediated cytotoxicity, HCC cells were identified by NK cells via MICA. As the Fc binds to its receptors on the surface of NK cells and macrophages, ADCC, CDC effects, and a longer half-life following engagement with neonatal receptors are all triggered (67). But shedding or downregulation of NKG2D ligands (NKG2DL) can prevent NKG2D activation, resulting in the escape of cancer cells from NKG2D-dependent immune surveillance.

There has been particularly little research into bispecific antibodies targeting NKp30 and NKp46 (68). CTX8573 is the first NKp30 × BCMA bispecific antibody that targets BCMA+ plasma cells and NK cells (69). The C-terminus of the antibody is attached to the anti-NKp30 Fab, and the mismatch is resolved using the same light chain. The Fc-terminus is given a de-fucose treatment to increase the impact on NK cells further. Intrinsic cells are effectively attracted to and activated by the binding of NKp30 and CD16A. Compared to monoclonal antibodies targeting CD16A, the ADCC potency is increased by more than 100 times using the NKp30 bispecific platform, which also maintains activity when CD16A is downregulated. Bispecific antibodies against NK redirection are still mainly used to treat hematological tumors. It is also unclear whether NK cells will enter solid tumors more readily than T cells for solid tumors. NK cells can produce relevant cytokines to attract other immune cells, which may further enhance the anti-tumor response. There is no doubt that strategies to increase the involvement of NK cells in anti-tumor response will be the future of tumor immunotherapy.

3.3 Bispecific antibody targeting immune checkpoint and co-stimulator for immune cell restoration

3.3.1 Immune checkpoint

Clinical cancer therapy approaches have undergone a revolutionary change due to recent discoveries on the roles of immune checkpoints in allowing cancers to avoid the innate/adapted immune system (7071). Immune checkpoint receptors of co-inhibitory molecules such as programmed cell death 1 (PD-1) and/or cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) are critical in maintaining self-tolerance and avoiding immune-mediated adverse effects on the host. However, numerous studies have shown that the (TILs) exhibit substantially elevated co-inhibitory receptors, which represent an exhausted phenotype and limited anti-tumor action (72). In addition, preclinical evidence suggests that T cell response has been inhabited on account of the upregulated expression level of programmed cell death 1 ligand 1 (PD-L1) on the surface of malignant cells to conducive tumor cell’s immune escape and limit the efficacy of anti-tumor immunotherapies (73). Immune checkpoint blockades (ICBs) have been established based on the mechanism mentioned above to break those negative regulators that prevent pre-existing anti-tumor immune responses from being activated. Some ICBs have shown notable efficacy in various cancers and have entered routine clinical implementation (74). Besides, A clinical trial reported the 5-year outcomes that nivolumab (anti-PD-1) combined with ipilimumab (anti-CTLA-4) among advanced melanoma patients has resulted in sustained long-term progression-free and overall survival (52%) compared with nivolumab group (44%) and ipilimumab group (26%) (75).

The therapeutic idea of blocking two inhibitory immune checkpoints has led to the rational design and development of bispecific antibodies that simultaneously target two inhibitory checkpoints expressed on the surface of the same or different cells. This has been made possible by the innovative success of ICBs immunotherapies and the improved clinical benefit rate observed in patients who have received combined treatment with ICBs. MGD019 is a monovalent investigational PD-1 × CTLA-4 bispecific DART compound designed to increase CTLA-4 checkpoint blockage in the TME based on a PD-1 binding mechanism. This single-molecule showed complete blockade of the PD-1/PD-L1 axis and Variable Inhibition of CTLA-4 in vitro and is well tolerated in non-human primates with increased T cell proliferation and expansion. Furthermore, the first-in-human study with MGD019 is ongoing in patients with multiple advanced solid tumors. After the dose-escalation phase, the analysis revealed acceptable safety and objective responses in various tumor types typically unresponsive to checkpoint inhibitor therapy (NCT03761017) (76). MEDI5752, fusing an anti–PD-1 mAb and the variable binding domains of Tremelimumab (anti-CTLA4) onto a DuetMab backbone, optimally designed with triple amino acid mutations of the human IgG1 constant heavy chain to reduce Fc-mediated immune effector functions. Dovedi, S.J. et al. discovered that this engineered molecule preferentially localizes and inhibits CTLA4 on PD-1+ T cells and rapidly induces internalization and degradation of PD-1.

As a result, the affinity for the CTLA4 receptor is markedly increased and saturated, increasing clinical benefit and minimizing further harm. In addition, current first-in-human research using MEDI5752 to treat advanced solid cancers showed promising partial responses with acceptable side effects (77). Different from MEDI5752, AK104 is an anti-PD-1/CTLA-4 bispecific antibody with the symmetrical structure of 4-valent IgG1-scfv developed by Akeso Biology. AK104 can rapidly mediate independent endocytosis of PD-1 or CTLA-4 based on good antigenic differentiated binding with high retention in tumor tissue. Recently, Cadonilimab (AK104) has been approved in China for treating patients with recurrent or metastatic cervical cancer who have failed prior platinum-containing chemotherapy.

Nowadays, most of these BsAbs target the next wave of inhibitory receptors expressed on TILs with one binding arm and block the PD-1/PD-L1 axis with the other binding arm to reverse acquired T cell exhaustion-driven resistance. Dual immunomodulator MGD013 targets LAG-3 and PD-1. Both target molecules are expressed on T cells following antigen stimulation. Based on the DART® form, MGD013 has been shown to effectively inhibit the binding of PD-1 to PD-L1 and PD-L2 while inhibiting the binding of LAG-3 and MHC II, which activate T cells by acting together. This bispecific antibody is in clinical phase I studies (NCT03219268). Similar preclinical dual immunomodulators include FS118 targeting PD-L1/LAG-3 and LY3415244 targeting PD-L1/TIM-3 (7879).

3.3.2 Co-stimulatory molecules

Co-stimulation assists the immune system in determining whether responses to antigenic stimuli and co-stimulatory receptors have been utilized for cancer immunotherapy. The targets of immune co-stimulation mainly focus on the B7-CD28 and TNFR family. In the B7-CD28 family, CD28 and ICOS are the main co-stimulatory receptors. OX40, CD40, CD27, 4-1BB, GITR, and CD30 belong to the TNFR family. CD28, one of the first identified co-stimulatory molecules constructively expressed on the surface of T cells, contributes to lowering the threshold, which is critical for TCR-mediated T cell activation and subsequently results in enhanced T cell proliferation, cytokine production, and release, as well as cell survival (80). Six healthy volunteers who participated in the first human clinical trial for TeGenero’s CD28 hyperagonist antibody TGN1412 had severe cytokine release syndrome and multiple organ failure. Because antibodies activate T cells even when the TCR is not involved, they create an immunological response that targets everyone.

Other co-stimulatory receptors are promising targets, transiently expressed on activated T cells via TCR-mediated signal identification rather than constituent expression like CD28. The affinity of agonistic antibodies for their targets must be optimized, not maximized. It is not the affinity but the intermittent exposure of co-stimulatory receptors that may become more crucial. Excitatory antibodies, though, are still in their infancy compared to immune checkpoint inhibitors. Utilizing excitatory antibodies alone might not be the best course of action. Co-stimulatory receptors play a unique role, and PD-1 therapeutic effectiveness depends on the CD28/B7 co-stimulatory pathway. PD-1 is widely believed to inhibit signal transduction through T-cell receptors (TCR). The study indicated that TCR co-stimulatory receptor CD28 is the primary target of PD-1 signal transduction (81). Lung cancer patients who responded to PD-1 therapy had more CD28+T cells, suggesting that CD28 may predict treatment response. Toxicity can be largely avoided by combining CD28 antibodies with another target.

TSA × CD28 bispecific antibodies have shown little or no toxicity in humanized immune system mice or primate models when used alone or combined with PD-1 antibodies (82). As a result, it might offer a safe “off-the-shelf” combination immunotherapy that could greatly improve anti-tumor effectiveness and trigger long-lasting anti-tumor immunity. Preclinical research on CD28 triple antibodies has recently demonstrated considerable promise, and this therapy is now widely recognized in theory. Sanofi created an anti-HER2, anti-CD3, and anti-CD28 tri-specific antibody to target, stimulate, and prolong the lifespan of T cells in malignancies (83).

The dissociation constants for HER2, CD28, and CD3 are 1.28, 1.0, and 1.43 nM, respectively. Each antigen arm shows a comparable affinity to the analogous single antigen arm in the presence of the other two antigens, demonstrating minimal interference between the various arms. The tri-specific antibody could promote tumor regression at low doses and achieve effective tumor suppression in both high and low HER2 expressing tumors. It was also found that CD4 cells, but not CD8 cells, were critical in promoting tumor growth arrest. The CD137 (4-1BB) is the most promising target in studies targeting co-stimulatory receptors. To reduce the toxicity of systemic CD137 agonists to the liver while maintaining efficacy, targeting CD137 with a bispecific molecule that binds to the tumor-associated antigen (TAA) and confining CD137+ T cell agonists to the tumor microenvironment appears to be an ideal approach. Targeting non-tumor toxic cytokine release syndrome is decreased by triggering only antigen-exposed T cells (CD137+ T cells). Independent of MHC, CD137 activation can expand tumor-reactive memory T cells. Additionally, CD137-targeted antibodies may be more resilient to antigen loss than CD3-targeted antibodies. Nevertheless, the clinical development of bispecific antibodies has been severely hampered by dose-dependent hepatotoxicity found in clinical studies with co-stimulatory molecules recognizing agonistic antibodies (8490).

Thus tumor-localized co-stimulatory bispecific antibodies have been developed to alleviate systemic toxicity after systemic effector T cell co-stimulation. PRS-343, engaging 4-1BB-specific Anticalin proteins to a modified variant of trastuzumab with a mutation modified IgG4 isotype to avoid the risk of ADCC and non–tumor-target activation of 4-1BB-positive lymphocytes, facilitates HER2+ tumor-localized co-stimulation of T cells with reduced peripheral toxicity (84). In addition, PRS-344/S095012, a synthesized tetravalent PD-L1/4-1BB bispecific antibody, showed stronger antitumoral activity and synergistic impact compared to the combination of mAbs via a tumor-localized 4-1BB-mediated activation (91). Preclinical models reflect that PRS-344/S095012-mediated 4-1BB activation depends on PD-L1, reducing the risk of peripheral toxicity and that 4-1BB co-stimulation occurs only in synchrony with TCR signaling, limiting its activity to antigen-specific T cells. Furthermore, DuoBody-PD-L1×4-1BB (GEN1046), the first-in-class bispecific immunotherapy agent, formed by the K409R and F405L mutations in the Fc CH3 region of two IgG1 antibodies and demonstrated pharmacodynamic immune effects and a manageable safety profile in a phase I trial of dose escalation in heavily pre-treated patients with multiple advanced refractory solid tumors (NCT03917381) (92).

In addition to bispecific antibodies, research for the CD28 and the 4-1BB target has been extended to triple and quadruple antibodies. However, the market for numerous distinct cancer antibody therapeutics is still in its infancy, and much research on its effectiveness and safety is still needed. Hepatotoxicity is being studied in the next generation of co-stimulation-targeted bispecific antibodies without compromising efficacy.

4 Bispecific antibody targeting non-immune cells in the TME for restricting tumor diffusion

The tumor microenvironment mainly consists of tumor cells and their surrounding immune and inflammatory cells, cancer-associated fibroblasts (CAFs), nearby mesenchymal tissue, microvasculature, and various cytokines and chemokines (Figure 2). It can be roughly divided into immune microenvironment based on immune cells and non-immune microenvironment. Angiogenesis, the process by which new blood vessels emerge from an already-existing vascular network, is crucial to tumor growth, progression, and metastasis (93). During the process, low oxygen tension (hypoxia) is a significantly important component of the TME driving tumor angiogenesis, which could upregulate multifarious proangiogenic growth factors like VEGF, placenta growth factor (PlGF), and angiopoietin 2 (Ang2) that correlate with the formation of new vessels through directly engaging in vessel growth (9495). VEGF family (VEGF-F) and Ang1-2/Tie-2 pathway are equally important in mediating tumor angiogenesis, and Ang-2 regulates vessel maturation in the later stage of angiogenesis, which contributes to promoting vascular formation with VEGF in the different stages. Upregulated levels of VEGF and Ang-2 demonstrated a worse prognosis factor in various tumor types (96), the blocking of the signaling pathway Ang-2 shows the effect of tumor growth inhibition with the decreased vascular formation. It normalizes remaining blood vessels with increased pericyte coverage (9799). Furthermore, the above blocking pathways have more significant effects when combined with anti-VEGFA drugs, even in certain tumor types with resistance (100102).

Figure 2
www.frontiersin.orgFIGURE 2 Bispecific antibodies exert anti-tumor effects in the immunosuppressive tumor microenvironment. In the complex tumor microenvironment, activated fibroblasts communicate with tumor cells, various inflammatory cells as well as stroma cells via secreting growth factors (TGFβ, VEGF, etc.) and other chemokines to provide potentially oncogenic signals and interact with the microvasculature, which induces an accelerated oncogenic extracellular-matrix microenvironment. BsAbs, aiming at blocking the interacting mechanism, transform the “cold” immune environment into the “hot” immune environment.

In addition to showing strong anti-tumor, antiangiogenic, and micrometastasis growth reducing effects in subcutaneous and orthotopic syngeneic mouse xenotransplantations, Ang-2-VEGF CrossMab also exhibits these effects in patient or cell line-derived humanized tumor xenografts with acceptable side effects compared to Ang-1 inhibition combined with anti-VEGF treatment on physiologic vessel growth (103). Furthermore, Kloepper, Riedemann et al. found that dual Ang-2/VEGF (CrossMab, A2V) antibody can prolong the survival of mice bearing orthotopic syngeneic (Gl261) GBMs or human (MGG8) GBM xenografts based on only VEGF pathway blocking failing to enhance overall survival of patients with GBM (104). The logical combination therapy of immune checkpoints and angiogenesis provides greater therapeutic effects, according to Schmittnaegel et al. On the other hand, increased intratumoral immune effector cell activation results in increased PD-L1 expression in tumoral endothelial cells (105).

The PD-L1 blockade could prolong the angiostatic effects of angiogenic factors receptor inhibition, enhancing vascular normalization to a certain extent. However, lacking tumor-infiltrating lymphocytes is related to primary resistance to ICIs. At the same time, dysfunctional tumor vasculature restricts lymphocyte T cell permeating into tumors, thus limiting the curative effect of immune-checkpoint blockade. Therefore, rational dual therapy modalities of anti-angiogenesis and immune checkpoint blocking like PD-1/PD-L1 signal pathways have broad clinical applicability (91105). The HB0025, with dual recognition of VEGFR and PD-L1 based on mAb-Trap technology, has shown enhanced anti-tumor benefits than either single drug treatment (106).

In addition to VEGF, the Notch pathway’s essential ligand delta-like ligand 4 (DLL4) plays a key role in tumor neo-angiogenesis and regulates the VEGF pathway’s signaling to prevent excessive vascularization (107). Due to severe target toxicities (such as hepatotoxicity and pulmonary hypertension) seen in the clinic, the DLL4 monoclonal antibody’s development has been stopped. The research strategy then shifted to bispecific antibodies for VEGF and DLL4. Navicixizumab (OMP-305B83), an IgG2 humanized BsAb, targets DLL4 and VEGF simultaneously. The data from the phase 1a study showed manageable toxicities and anti-tumor activity in various tumor types, which encouraged an ongoing phase 1b clinical trial to further assess the curative effect in pre-treated ovarian cancer patients with platinum resistance (NCT03030287) (108). Besides, ABL001 (VEGF × DLL4), with enhanced biological anti-tumor activity in xenograft models than VEGF or DLL4 monoclonal therapeutic antibodies alone, is under phase 1 clinical study to evaluate combination therapy effect with heavy chemotherapy (NCT03292783) (92).

Cancer-associated fibroblasts (CAFs), ranking in the stromal cell population, which compose of diverse subpopulations with distinct functions in cancer, represent the most considerable component of the tumor microenvironment (TME) (109). Abundant studies have confirmed that CAF populations could exert different but mutual functions modulating tumor growth, proliferation, tumor metastatic dissemination, and extracellular matrix components remodeling, simultaneously correlated to immunosuppression TME establishment and chemoradiotherapy resistance (109113). Recently, an in-depth study on the crucial role that CAFs play in the tumor immune microenvironment’s (TIME) pro-oncogenic functions has been done, showing CAFs as a promising therapeutic target (114). Fibroblast Activation Protein (FAP), a marker expressed on the surface of CAFs and detected in various cancer types of poor prognosis, has appeared as a novel strategy for targeted immunotherapies. Bispecific FAP-targeted 4-1BB ligand (RG7826), correctly assembled through CH1-CL domain crossover, knob into hole (KIH) amino acid mutation in the fragment crystallizable (Fc) domain, as well as mutations in CH1 (EE) and CL (RK) (12115), led to intensive IFN-γ and granzyme B secretion in human tumor samples while combined with tumor antigen-targeted (CEA) T cell bispecific (TCB) molecules (89116). A new FAP-DR5 (death receptor 5) tetravalent bispecific antibody called RG7386 aims to activate extrinsic DR5. In preclinical patient-derived xenograft models, hyperclustering dependent on the tumor cells’ apoptotic pathway and binding to FAP-positive stroma led to durable tumor reduction, which is currently being assessed in phase-I clinical research (117118).

5 Bispecific antibody changing TGF-β signal pathway to improve the tumor microenvironment

Transforming growth factor (TGF)-β is a multifunctional cytokine that plays a dual role, tumor suppressor or promoter, in a cellular or context-dependent manner, known as the TGF-β “paradox”. In early-stage tumors, the TGF-β pathway induces apoptosis and inhibits tumor cell proliferation. In contrast, it has a tumor-promoting role in advanced stages by regulating genomic instability, epithelial-mesenchymal transition (EMT), neoangiogenesis, immune evasion, and cell metastasis (Figure 2). Previous research suggests that response rates to TGF-β monoclonal antibody therapy are low, which could be related to the fact that it is not a tumor promoter. As a result, one of the primary avenues of advancement in this sector has been combination therapies, which include combinations with ICIs (e.g., PD-1/PD-L1 antibodies), cytotoxic drugs, radiotherapy, cancer vaccines, and so on.

The discovery that TGF-β antibody induces potent anti-tumor immunity when combined with PD-L1 antibodies was made in a study of patients with uroepithelial carcinoma who metastasized after receiving PD-L1 antibodies (119). In this study, CD8+ T cells were found in the patients’ tumor interstitium containing fibroblasts and collagen but not in the tumor interior, TGF-β signal limits T cell infiltration. At the same time, some researchers fused TGF-β receptor II and PD-L1 antibodies into a tetravalent BsAb, M7824, and found that the bispecific antibody had better anti-tumor effects than the monotherapies in homozygous mouse models of breast and colon carcinoma. The Phase I clinical study (NCT03917381) of M7824 patients with non-small cell lung cancer (NSCLC) evaluated its efficacy and safety. The median follow-up was 51.9 weeks, with an objective remission rate (ORR) of 21.3%, partial remission (PR) of 21.3%, stable disease (SD) of 16.3%, and disease progression (PD) of 48.8% for all patients; disease control rate (DCR) of 40%; overall survival (OS) of 13.6 months; median progression-free survival (PFS) of 2.6 months; The 12-month PFS rate was 20.1%; median duration of remission (DOR) was 14.1 months. A dose of 1200 mg was determined to be the recommended dose for the Phase II study. But German Merck and collaborator Glaxo finally announced that a Phase III (INTR@PID Lung 037) interim analysis of bintrafusp alfa (M7824) showed that it could not outperform PD-1 antibody Keytruda. Bintrafusp alfa was then stopped as a single-agent second-line treatment for locally progressed or metastatic biliary tract carcinoma (BTC) in Phase II INTR@PID BTC 047 due to failure to reach the primary endpoint. Bintrafusp alfa has failed four clinical trials in a row since 2021. M7824 is also being tested in various indications, including esophageal, biliary tract, and gastric cancers. With the discovery that transforming growth factor-β (TGF-β) inhibits T helper cell (Th2)-mediated cancer immunity, researchers constructed a bispecific antibody targeting CD4 and TGF-β, 4T-Trap, which selectively inhibits TGF-β signaling by CD4+ T cells in lymph nodes (120121), leading to cancer cell apoptosis and vascular rearrangement. Due to TGF-β multifunctionality and the potential for major side effects from the total blockade, inhibition of TGF-β in cancer therapy has not been successful. However, 4T-Trap focuses TGF-β blocking compounds directly on CD4+ T cells to minimize adverse effects. And combining 4T-Trap with a VEGF inhibitor may help to prevent the spread of vascular-mediated malignancy.

Due to the diversity of TGF-β signaling, combinations are likely to be effective only when TGF-β is the tumor-promoting signal. The efficacy of anti-TGF-β must be carefully analyzed when TGF-β exerts tumor-suppressive effects or when the receptor for TGF-β is mutated. In addition, the combination of drugs may cause a strong immune response in patients; whether the patient can tolerate it and the deepening of side effects is also a question worthy of consideration. A deeper understanding of the communication and interactions between the various components of the tumor patient’s organism and TGF-β signaling is key to improving clinical efficacy.

6 Approaches to avoid on-target off-tumor adverse effects

A reinvigorated anti-tumor immunity can be a double-edged weapon while many programs explore alternative modes of action correlated with bispecific antibodies’ target pathways. The efficacy of genetically modified bispecific antibodies against cancer has increased greatly at the expense of improved toxicities in normal tissues and systemic cytokine release immune responses (122123). Strategies of conditionally activating T cells within tumors and modifying target affinities to mitigate or conquer the “on-target off-tumor” adverse effect of bispecific antibodies have been taken into consideration and investigation (52124126).

To avoid T cell autoreactivity to normal target-expressing tissues while there is generally lacking tumor-specific targets in solid tumors, Slaga, D., and colleagues have designed and exploited a T cell-dependent bispecific (TDB) antibody with a bivalent low-affinity HER2 recognition binding domain which can selectively target HER2-overexpressing tumor cells from normal human tissues with low amounts of HER2 expressing (52). Besides, antibody binding affinity is a major factor for overall tolerability, while the higher affinity for CD3 is related to rapidly increasing peripheral cytokine concentrations. While having no impact on anti-tumor effectiveness, anti-HER2/CD3 TDBs with lower CD3 binding affinity is better tolerated in vivo. Higher HER2 affinity aids in tumor-killing action but also causes more severe toxicity, including cytokine release syndrome, in HER2-expressing tissues. a dose-fractionation technique, which offers an application strategy for the affinities-modulated antibodies, has been used to address such a problem (124).

Disorganized tumor tissue growth and rapid cell division contribute to complicated extracellular features of the TME, such as a hypoxia environment with low pH, increased extracellular matrix remodeling, and upregulated proteolysis, which has contributed to the exploration of conditionally activating T cells in the TME based on the preferentially binding of BsAbs at hypoxic extracellular conditions as well as local liberation of the BsAbs antigen-binding sites released by tumor-associated proteases (126135). Preclinical research has shown that T cell recruiting BsAbs (TCBs) conditionally activated by intratumorally proteolytic cleavage can prolong therapeutic windows, successfully avoid dose-limiting toxicities, and significantly prolong tumor regression (131133). Furthermore, Geiger, M. and colleagues have generated a protease cleavable activated anti-folate receptor 1 TCB (Prot-FOLR1-TCB) via masking the anti-CD3 binding domain with an anti-idiotypic anti-CD3 scFv N-terminally connected to the anti-CD3 variable heavy chain through a protease cleavable linker, which has shown validly releasing of the anti-CD3 binding moiety in active proteases enriched tumor microenvironment, thus effectively reducing potential on-target toxicity through sparing normal tissues with low degrees FOLR1 expression while mediating efficient anti-tumor ability in FOLR1-positive tumor tissue (126). Besides, binding sites of the BsAb assembling intratumorally from two half-molecules is an ideal approach to further increasing tumor selectivity (125136).

7 Conclusion and prospect

As a result of the FDA’s approval of Blinatumomab for the treatment of recurrent ALL and the ongoing need for BsAbs, novel formats aimed at enhancing therapeutic efficacy and safety have been developing for the treatment of solid tumors as well as hematologic malignancies. BsAbs have gained momentum over the past decade. Despite promising progress in the clinical application field of bispecific immunomodulatory antibodies in part of human tumor types, more prominent anti-tumor efficacy in most solid tumors still needs constant exploration. Furthermore, dozens of BsAbs with different targets combinations have exhibited potent anti-tumor effect in preclinical studies, but most of the positive preclinical outcomes could not be further validated in the clinic. With increasingly diverse BsAbs entering preclinical and clinical trials, various challenges have emerged hampering the development of BsAbs. The development of BsAbs is dimensionally more difficult than that of a monoclonal antibody. Selecting the optimal targets combination is only the first step, followed by the right choosing of a rational format and designing the molecule according to the targets as well as the biology of the diseases. Besides, inappropriate clinical design and administration regimens will expose patients into significantly higher toxicities, which can be avoided via optimization of treatment strategies, dosage, timing, and sequence to some extent. We anticipate that more comprehensive exploration in the field of bispecific immunomodulatory antibodies will broaden the prospect of cancer immunotherapy.

Author contributions

All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.


This work was supported by 1.3.5 Project for Disciplines of Excellence, West China Hospital, Sichuan University (Grant No. ZYJC21043), the National Natural Science Foundation of China (31971390), and Sichuan Science and Technology Program (2021YFH0142).

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of insterest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

RAbD is a new biotechnology company founded by Fox  Chase Cancer Center investigators Gregory Adams, Ph.D., Matthew Robinson, Ph.D. and Roland Dunbrack, Ph.D. that is focused on the knowledge-based design of antibodies that bind to key functional, often highly conserved and difficult to target epitopes. We are using homology modeling, crystal structures, protein docking and design software and algorithms to drive combinatorial sampling of CDRs to computationally design new antibodies and then express, validate and perform further design in an iterative manner.Brian Smith, Ph.D., MBA is RAbD Biotech’s Business Development Lead.

Contact information for RAbD Biotech:

Website  http://rabdbiotech.com/


Twitter @RAbDBiotech

The overall goal of RAbD is to

“drug the undruggable”

The company using in silico design methods to design to produce novel antibodies and biomimetics. The company is developing a first in class biomimetic, RaD-003, for the treatment of ovarian cancer.  Ovarian  cancer is one of the most deadly of all women’s cancers, with very low 5 year survival rates.  An expected 22,000 US women a year will be diagnosed and expected 16,000 will die every year.  Cisplatin/paclitaxel therapy is only approved and effective chemotherapy for ovarian cancer yet resistance develops quickly and is common. RaD-003  targets the MISII receptor (Mullerian Inhibiting Substance Type II Receptor), which is expressed on ovarian cancer cells but not on normal ovarian epithelium.

It has been shown that activation of this receptor by the Mullerian Inhibiting Substance (MIS) has antitumor activity in ovarian cancer.

The MISII receptor had been considered undruggable as

  • MIS is too expensive and difficult to produce
  • previous attempts to develop therapeutic antibodies ot MISIIR have proven difficult

Therefore, the company used their computational platform to produce a “first in class” chimeric biomimetic to more effectively target and activate MISIIR.

For  more information about this meeting and the Mid-Atlantic Bioangels and 1st Pitch please see posting on this site

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Report on the Fall Mid-Atlantic Society of Toxicology Meeting “Reproductive Toxicology of Biologics: Challenges and Considerations.  Author, Reporter: Stephen J. Williams, Ph.D.

The fall 2012 Meeting of the Mid-Atlantic Society of Toxicology (MASOT) focused on the challenges and solutions in developing proper Development and Reproductive Toxicology (DART) studies with regards to the newer classes of bio-therapeutics such as vaccines, antibody-based therapies, and viral-based therapies.  The full meeting and MASOT links can be found at http://www.masot.org.   The overall synopsis of the meeting talks agreed, that although the general aim and design of DART studies for biological are very similar to DART studies for small molecule therapeutics, it is more necessary to take into consideration the pharmacodynamics, pharmacokinetic differences between biologics and small molecules.   In addition it is imperative to use pharmacologically-relevant species, such as non-rodent (guinea pig and non-human primate). The meeting was highlighted by the keynote speaker, Dr. A. Wallace Hayes, renowned board-certified toxicologist, committee and expert panel member for National Academy of Sciences, NIEHS, EPA and Department of Defense, and editor of well-known textbooks including Principles and Methods of Toxicology.  Dr. Hayes discussed a timeline of milestones in the field of toxicology.

The following are the meeting talk abstracts as well as notes for each presenter.

What’s So Different About DART Assessment of Biologics? Christopher Bowman Ph.D., DABT (Pfizer, Inc.)

Abstract:  The aim of developmental and reproductive toxicity (DART) safety assessment of a biologic is no different from that of a small molecule. Both cases consist of evaluating the potential for maternal toxicity, pre- and postnatal development toxicity (including juvenile toxicity) and effects of fertility (reproduction).  The differences lie in the in the product attributes of a specific biologic, the pharmacological response, the potential for undesirable toxicities and how these product attributes influence and are influenced by the biology.  Thus the primary challenge for developing a DART strategy for a biologic are derived from the complexities of these biomolecules and how that dictates a case-by-case strategy for appropriately evaluating the potential for developmental and reproductive toxicity. Most protein biologics have very limited potential for off-target toxicities, but this is not necessarily the case for other modalities such as anti-sense oligonucleotides and antibody-drug-conjugates.  In these cases, off-target toxicities can be a major feature of the DART safety assessment.  The most noticeable difference in DART assessment of biologics is the need to conduct these studies in pharmacologically relevant species and how that can influence the overall nonclinical strategy (including DART).  This has led to increased use of non-human primates as a model system and led to optimizations of this model for this purpose and revisions to international guidelines.

Notes:   Dr. Bowman emphasized the need to understand the type of biological you are testing and to both devise DART studies based on this information, additional endpoint you may want, as well as carefully choosing the correct species most relevant to the biologic.  He highlighted general differences between small molecules versus a biologic with respect to their pharmacology.  These differences are summarized in the Table below:

  Small Molecule Biologic-based therapy
Species specificity Low High
Route of administration Usually oral Parental
ADME (PK, bio-distribution etc.) Wide distribution Low distribution

He noted that clinical trials for biologics rarely include reproductive toxicity so the preclinical DART study is of utmost importance.  He also emphasized that currently, the FDA requires two species for DART testing of small molecule therapies (usually one rodent and one non-rodent).  However this is not possible with many biologics as species is to be taken in consideration when designing a meaningful DART study.  Study designs can be like most DART studies but want to have a steady exposure during fetal organogenesis, use high doses (10 times the clinical dose) to achieve maximal pharmacology, confirm exposure to fetus and to F1 generation, and determine embryolethality.  Some biologics like interferon and insulin-growth factor receptor (IGFR) antagonists are fetal abortifactants. In fact Lucentis (Ranibizumab) and Macugen (Pegaptanib) were approved with no or little DART studies, however these drugs showed reproductive toxicity, resulting in warning concerning pregnancy on the label. Also important is the effect on the immune system and reproductive system of offspring, as well as the pharmacodynamics profile in the offspring.

Species Selection for Reproductive and Developmental Toxicity Testing of Biologics; Elise M. Lewis, Ph.D. (Charles River Preclinical Services)

Abstract:  Regulatory guidelines for developmental and reproductive toxicology studies require selection of “relevant” animal models as determined by kinetic, pharmacological, and preceding toxicological data.  Rats, mice, and rabbits are the preferred animal models for these studies based on historical experience and well-established procedures and study protocols.  However, due to species specificity and immunogenicity issues, developmental and reproductive toxicology testing for biologics is limited to a pharmacologically relevant animal model as described in the ICH s6 guideline.

Notes:  Dr. Lewis notes that DART studies in guinea pigs and hamsters represent a cost effective alternative to large animal models as well as the benefit of shorter duration and ability to assess mating behavior.  She also notes that reproductive toxicology of vaccines should be done in an animal model that can elicit an immune-response to the vaccine, especially to determine any maternal-fetal interaction.  For example, a vaccine may be directed to a maternal protein which when suppressed, may negatively impact the developing fetus.  However it is important to remember that guinea pigs can spontaneously abort so it is good to have proper control arms of a substantial size in order to statistically determine the impact of those spontaneous abortions.



Placental Transfer of an Adnectin Protein During Organogenesis in Guinea Pigs Using a Radiolabeled Methodology; Lakshmi Sivaraman, Ph.D. (Bristol-Myers Squibb)

Abstract:  Knowledge regarding the placental transfer of large molecular weight therapeutics is important to support the enrollment of women of childbearing potential in clinical trials.  There is limited information in the scientific literature that reports the extent to which the conceptus is exposed to these large molecules during organogenesis.  Placental transfer of large therapeutics has been difficult to quantify, due to limited blood volumes that can be obtained from the embryo, as well as insufficient assay sensitivity.  Thus, it is possible that embryos are exposed to pharmacologically active concentrations after maternal drug exposure. We have adopted a radiolabeled approach to quantitate embryo-fetal exposure of a novel protein therapeutic platform (adnectins). Adnectins are fibronectin-based proteins containing domains engineered to bind to targets of therapeutic interests.

Notes: Adnectins molecular weight is typically less than monoclonal antibodies and while IgG is not transferred in great quantity past the placental barrier there have been studies in human indicating maternal-fetal transfer of monoclonal antibodies.  This is particularly important for two reasons:  the monoclonal interacts with a target important in development, or the fetal immune system could be augmented.  Their work will be published in Drug Metabolism and Disposition.  In general Dr. Siveraman engineered a radiolabel on adnectin and used different detection methods to quantify the fetal exposure to a single maternal dose.  Dr. Siverman was able to detect radiolabel in the fetus however it is not clear whether this is a significant amount.

Reproductive Toxicity Testing for Biological Products in Nonhuman Primates: Evolution and Current Perspectives: Gary J. Chellman, Ph.D., DABT (Charles River Preclinical Services)

Notes:  Dr. Chellman gave a review of the current trends being driven by regulatory agencies with regard to nonhuman primate DART studies of biopharmaceuticals.  He noted that an advantage using nonhuman primates were the close physiologic resemblance to humans and because a large animal could monitor pregnancy over time using ultrasound technology.  In general, Dr. Chellman spoke about new study designs which not only reduce the number of animals required but also significantly reduce costs.  For example, a DART study which cost upward of $750,000 now can be done for as little as $350,000.  Dr. Kary Thompson of Bristol Myers Squibb then gave a talk about use of these new enhanced designs to determine reproductive toxicity issues with ipilimumab (Yervoy).

Other research papers on Pharmaceutical Intelligence and Reproductive Biology, Bio Insrumentation, Endocrinology Genetics were published on this Scientific Web site as follows

Non-small Cell Lung Cancer drugs – where does the Future lie?

Reboot evidence-based medicine and reconsider the randomized, placebo-controlled clinical trial

Every sperm is sacred: Sequencing DNA from individual cells vs “humans as a whole.”

Leptin and Puberty

Gene Trap Mutagenesis in Reproductive Research

Genes involved in Male Fertility and Sperm-egg Binding

Hope for Male Contraception: A small molecule that inhibits a protein important for chromatin organization can cause reversible sterility in male mice

Pregnancy with a Leptin-Receptor Mutation

The contribution of comparative genomic hybridization in reproductive medicine

Sperm collide and crawl the walls in chaotic journey to the ovum

Impact of evolutionary selection on functional regions: The imprint of evolutionary selection on ENCODE regulatory elements is manifested between species and within human populations

Biosimilars: CMC Issues and Regulatory Requirements

Biosimilars: Intellectual Property Creation and Protection by Pioneer and by Biosimilar Manufacturers

Assisted Reproductive Technology Cycles and Cumulative Birth Rates

Innovations in Bio instrumentation in Reproductive Clinical and Male Fertility Labs in the US

Increased risks of obesity and cancer, Decreased risk of type 2 diabetes: The role of Tumor-suppressor phosphatase and tensin homologue (PTEN)

Guidelines for the welfare and use of animals in cancer research

Every sperm is sacred: Sequencing DNA from individual cells vs “humans as a whole.”



Read Full Post »

Curator: Aviva Lev-Ari, PhD, RN

For IP and Legal aspects of Biosimilars, go to:

Biosimilars: Intellectual Property Creation and Protection by Pioneer and by Biosimilar Manufacturers


For CMC and Regulatory Affairs of Biosimilars, go to:

Biosimilars: CMC Issues and Regulatory Requirements


The patent provisions of the Biosimilar Act, 2009 establish demanding and time-sensitive disclosure requirements. ObamaCare upheld by the Supreme Court is a victory for future development of pathways for biosimilar regulatory approval and eventually biosimilar generic drugs.

With the upheld ObamaCare, critical parts of the PPACA constitutional, and with it the BPCIA giving the FDA authority to approve biosimilars.

Had the PPACA been stricken in part or in its entirety, it would have presented obstacles to the BPCIA surviving in its present form. The US government has been critical of the 12-year data exclusivity period for Pioneer Innovators, calling for it to be shortened to 7 years (12 years is favorable to Pioneer Innovators and less favorable for Biosimilar manufacturers). The upheld ObamaCare, PPACA and BPCIA, constitutional, has prevented a multiyear delay in biosimilar approval. Thus, it was the best scenario for the biologics industry.

Thus, projection of Sales for Biosmilars as % of top 100 U.S. Pharmaceutical will receive a special meaning and an expected enhanced market share for 2012 year end and beyond 2012.

Biosimilars are occupying the Following ranking in the U.S. Pharmacuetical Sales – 2012: Top 100 Drugs for Q1 2012 by Sales: 10, 11, 12 13, 15, 24, 27, 29, 33, 35, 39, 57, 58, 62, 65, 70,  72, 74, 90, 98, 99. In addition the following biosimilars did not make the Top 100 list:

Biosimilar Drugs by US Sales – not included in the Top 100 Drug List 

Recombinate $2.9 1998 — Antihemophilic Factor VIII (Recombinant) by Baxter 5.7 Billion in 2012

Cerezyme $1.5 1994 —  Gaucher disease and Fabrazyme for Fabry disease by Genzyme 200 millions in sales

TYSABRI(R) (natalizumab) revenues were $280 million, in-line with the second quarter of 2011 by Elan and Biogen

NovoSeven $1.4 1999 —  Anti-fibrinolytics by Novo Nordisk – $1.5Billion

Synagis $1.3 1998 — Generic Name:  palivizumab     Anti-virals by AstraZeneca  $570 millions

Humulin $1.1 1992 Insulin Human by Eli Lilly $ 1.2 Billion

Kogenate FS $1.1 1993 — octocog alfa    Anti-fibrinolytics By Bayer $1.4 billion

U.S. Pharmacuetical Sales – 2012: Top 100 Drugs for Q1 2012 by Sales – Small Molecule Drugs (in green) and Biosimilars (in red)

The following is a list of the top 100 pharmaceutical drugs by retail sales in 2012, listed by U.S. sales value and drug name. Last updated: July 2012 (updated quarterly)


Rank Drug

Sales ($000)

1 PlavixBristol-Myers Squibb Company



2 NexiumAstraZeneca Pharmaceuticals



3 AbilifyOtsuka Pharmaceutical Co.



4 SingulairMerck & Co., Inc.



5 SeroquelAstraZeneca Pharmaceuticals



6 Advair DiskusGlaxoSmithKline



7 CrestorAstraZeneca Pharmaceuticals



8 CymbaltaEli Lilly and Company



9 atorvastatinGeneric Drug



10 HumiraAbbott Laboratories




11 RemicadeCentocor Ortho Biotech, Inc




12 EnbrelAmgen Inc.




13 NeulastaAmgen Inc.




14 LipitorPfizer Inc



15 RituxanGenentech, Inc




16 CopaxoneTeva Pharmaceuticals



17 AtriplaGilead Sciences, Inc.



18 OxyContin



19 SpirivaBoehringer Ingelheim Pharmaceuticals, Inc



20 AvastinGenentech, Inc



21 ActosTakeda Pharmaceuticals North America, Inc



22 JanuviaMerck & Co., Inc.



23 TruvadaGilead Sciences, Inc.



24 LantusSanofi-Aventis



25 DiovanNovartis Corporation



26 LexaproForest Pharmaceuticals, Inc



27 EpogenAmgen Inc.




28 LyricaPfizer Inc



29 Lantus SolostarSanofi-Aventis




30 enoxaparinGeneric Drug



31 EloxatinSanofi-Aventis



32 CelebrexPfizer Inc



33 HerceptinGenentech, Inc




34 Diovan HCTNovartis Corporation



35 LucentisGenentech, Inc




36 SynagisMedImmune, Inc



37 NamendaForest Pharmaceuticals, Inc



38 GleevecNovartis Corporation



39 AvonexBiogen Idec




40 VyvanseShire US Inc



41 olanzapineGeneric Drug



42 IncivekVertex Pharmaceuticals



43 One Touch Ultra



44 SuboxoneReckitt Benckiser Pharmaceuticals Inc.



45 methylphenidateGeneric Drug



46 ZetiaMerck & Co., Inc.



47 AndroGelAbbott Laboratories



48 ProvigilCephalon, Inc.



49 LidodermEndo Pharmaceuticals



50 TriCorAbbott Laboratories



51 SymbicortAstraZeneca Pharmaceuticals



52 CombiventBoehringer Ingelheim Pharmaceuticals, Inc



53 ProAir HFATeva Pharmaceuticals



54 Seroquel XRAstraZeneca Pharmaceuticals



55 amphetamine/dextroamphetamineGeneric Drug



56 NasonexMerck & Co., Inc.



57 NovologNovo Nordisk Inc.




58 ProcritJanssen Pharmaceuticals, Inc




59 AlimtaEli Lilly and Company



60 ViagraPfizer Inc



61 GeodonPfizer Inc



62 Rebif




63 budesonideGeneric Drug



64 NiaspanAbbott Laboratories



65 HumalogEli Lilly and Company




66 Flovent HFAGlaxoSmithKline



67 LovazaGlaxoSmithKline



68 LevemirNovo Nordisk Inc.



69 Adderall XRShire US Inc



70 NeupogenAmgen Inc.




71 ReyatazBristol-Myers Squibb Company



72 AranespAmgen Inc.




73 metoprololGeneric Drug



74 NovoLog FlexPenNovo Nordisk Inc.




75 VytorinMerck & Co., Inc.



76 JanumetMerck & Co., Inc.



77 IsentressMerck & Co., Inc.



78 escitalopramGeneric Drug



79 CialisEli Lilly and Company



80 AciphexEisai Corporation



81 PradaxaBoehringer Ingelheim Pharmaceuticals, Inc



82 SolodynMedicis Pharmaceutical Corporation



83 fentanylGeneric Drug



84 ZyprexaEli Lilly and Company



85 VelcadeTakeda Pharmaceuticals North America, Inc



86 RestasisAllergan, Inc



87 LunestaSunovion Pharmaceuticals Inc.



88 acetaminophen/hydrocodoneGeneric Drug



89 PrezistaJanssen Pharmaceuticals, Inc



90 PegasysGenentech, Inc




91 ZyvoxPfizer Inc



92 Prevnar 13Wyeth



93 LovenoxSanofi-Aventis



94 BenicarDaiichi Sankyo



95 VESIcareAstellas Pharma US



96 Ventolin HFAGlaxoSmithKline



97 OrenciaBristol-Myers Squibb Company



98 BetaseronBayer Healthcare Pharmaceuticals




99 ErbituxBristol-Myers Squibb Company




100 DexilantTakeda Pharmaceuticals North America, Inc



Source: IMS Health (Midas).

Biosimilars Drugs by US Sales – not included in the Top 100 Drug List 

Recombinate $2.9 1998 — Antihemophilic Factor VIII (Recombinant) by Baxter 5.7 Billion in 2012

Cerezyme $1.5 1994 —  Gaucher disease and Fabrazyme for Fabry disease by Genzyme 200 millions in sales

TYSABRI(R) (natalizumab) revenues were $280 million, in-line with the second quarter of 2011 by Elan and Biogen

NovoSeven $1.4 1999 —  Anti-fibrinolytics by Novo Nordisk – $1.5Billion

Synagis $1.3 1998 — Generic Name:  palivizumab     Anti-virals by AstraZeneca  $570 millions

Humulin $1.1 1992 Insulin Human by Eli Lilly $ 1.2 Billion

Kogenate FS $1.1 1993 — octocog alfa    Anti-fibrinolytics By Bayer $1.4 billion

2011 US Sales vs. 2008 US Sales (in Billions) for Top Selling Biologics

Source for 2008 Sales


Source for 20011, Q1 2012 Sales


Drug Name,  2008 Sales, Year approved , Indication

[i.e. Drug Name Enbrel,  2008 Sales $8.0B, Year approved 1998 , Indication RA]

Enbrel $8.0 1998 — RA, psoriatic arthritis, or ankylosing spondylitis indication

Q1 2012 12 (1) $890,135 1.92% 823 -4.63%
Q4 2011 11 (1) $873,343 1.67% 863 1.77%
Q3 2011 12 (1) $858,997 1.27% 848 -2.97%
Q2 2011 13 (2) $848,230 3.77% 874 3.19%
Q1 2011 11 $817,401 847


Remicade $7.9 1998 — RA & Chron’s Disease

Q1 2012 11 (2) $899,453 10.04% 1,556 10.04%
Q4 2011 13 (3) $817,365 -7.02% 1,414 -9.82%
Q3 2011 10 $879,054 1.52% 1,568 1.03%
Q2 2011 10 (2) $865,903 7.61% 1,552 7.11%
Q1 2011 12 $804,699 1,449


Humira $7.3 2002  — treat rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, and plaque psoriasis

Q1 2012 10 $928,124 2.50% 546 -2.85%
Q4 2011 10 (1) $905,527 3.18% 562 2.55%
Q3 2011 11 (3) $877,641 3.95% 548 3.01%
Q2 2011 14 $844,296 6.32% 532 2.31%
Q1 2011 14 $794,076 520


Rituxan $7.3 1997 — cancer medicines to treat non-Hodgkin’s lymphoma or chronic lymphocytic leukemia.

Q1 2012 15 (1) $756,875 -1.91% 547 -0.91%
Q4 2011 14 (2) $771,622 6.96% 552 4.74%
Q3 2011 16 $721,408 -1.77% 527 -1.86%
Q2 2011 16 (4) $734,378 7.26% 537 5.09%
Q1 2011 20 $684,666


Second Quarter 2012 Highlights: RITUXAN(R) (rituximab) revenues from our unconsolidated joint business arrangement were $285 million for the quarter, an increase of 31% year-over-year. As previously disclosed, during the second quarter of 2011 our share of RITUXAN revenues from unconsolidated joint business was reduced by approximately $50 million to reflect our share of damages and interest that might be awarded in relation to an intermediate decision in Genentech, Inc.’s ongoing arbitration with Hoechst GmbH


Herceptin $5.7 1998 —  treat metastatic breast cancer that has progressed after treatment with other chemotherapy

Q1 2012 33 $425,687 -0.06% 155
Q4 2011 33 (2) $425,931 7.61% 155 4.73%
Q3 2011 31 (1) $395,804 -0.64% 148 -0.67%
Q2 2011 32 (4) $398,348 3.62% 149 1.36%
Q1 2011 36 $384,428 147


Lantus $5.1 2000 — long-acting form of the hormone insulin.

Q1 2012 29 (5) $448,388 9.81% 3,737 7.32%
Q4 2011 34 $408,336 8.54% 3,482 7.07%
Q3 2011 34 (2) $376,208 4.53% 3,252 6.00%
Q2 2011 36 (5) $359,907 7.80% 3,068 8.30%
Q1 2011 41 $333,878 2,833


Epogen/Procrit $5.1 1989Anemia, low RBC

Worldwide, sales of the two drugs – sold under the brand names Epogen, Procrit and Aranesp – exceeded $9 billion in 2005 for Amgen and Johnson & Johnson, their makers.  Johnson & Johnson, which sells epoetin under the brand names Procrit in the United States and Eprex everywhere else, reported sales of $2.4 billion in the first nine months of 2006, down slightly from 2005.

Amgen Recalls Anemia Medications for Glass Fragments09/24/2010 – Drug-makers Amgen (AMGN) and Johnson & Johnson (JNJ) are voluntarily recalling two brandsof an injectable anemia medication because vials containing the drug may have tiny glass flakes. The drug, Epoetin alfa, is marketed under the brand names Epogen and Procrit.Known as lamellae, the glass fragments are created by the interaction of the drug with glass vials during storage, Amgen said in a statement announcing the recall. The recall is being conducted in cooperation with the Food and Drug Administration, Amgen said.

Latest study shows anemia drugs Epogen, Aranesp and Procrit cause strokes, says FDA

Posted on January 7, 2010

Anemia drugs sold by Amgen and Johnson & Johnson have been reported to cause strokes when prescribed in high doses, according to an article from the FDA, recently published in the The New England Journal of Medicine. The law firm of Aylstock, Witkin, Kreis & Overholtz is investigating the FDA’s recent announcement.

The FDA commentary said the latest study and previous studies “raise major concerns” about the use of these drugs to treat anemia caused by kidney disease. The drugs are also used to treat anemia caused by chemotherapy. Studies over the past several years have revealed a link between the drugs and heart attacks, strokes, and other problems.

Amgen’s anemia drugs include Epogen and Aranesp. Johnson & Johnson sells anemia drug Procrit, which is produced by Amgen. The drugs are designed to raise red blood cell levels, to promote delivery of oxygen to body tissues.


Epogen / Procrit / Aranesp: The July 2012 News Report Which Tells Story Of Big Pharma Profits Over Patient Safety And Drug Efficacy

Once The FDA Started Paying Attention The Writing On The Wall Became Apparent, Albeit Too Late For Some

(Posted by  at DrugInjuryWatch.com)

This lengthy and well-presented news report, “Anemia drugs made billions, but at what cost?”, written by Peter Whoriskey and published July 19, 2012 by The Washington Post (free registration required), is a must-read for anyone with a concern or interest in how larger pharmaceutical companies might put corporate profits ahead of patient safety and drug efficacy.

Here is an excerpt from this Washington Post article which will give you a sense of what went on that, in hindsight, is so disturbing:

For years, a trio of anemia drugs known as Epogen, Procrit and Aranesp ranked among the best-selling prescription drugs in the United States, generating more than $8 billion a year for two companies, Amgen and Johnson & Johnson. Even compared with other pharmaceutical successes, they were superstars. For several years, Epogen ranked as the single costliest medicine under Medicare: U.S. taxpayers put up as much as $3 billion a year for the drugs.

The trouble, as a growing body of research has shown, is that for about two decades, the benefits of the drug — including “life satisfaction and happiness” according to the FDA-approved label — were wildly overstated, and potentially lethal side effects, such as cancer and strokes, were overlooked.

Last year, Medicare researchers issued an 84-page study declaring that among most kidney patients, the original and largest market for the drugs, there was no solid evidence that they made people feel better, improved their survival or had any “clinical benefit” besides elevating a statistic for red blood cell count.

As for some of the key events which led up to this revelation of sorts, we start with a June 24, 2011 FDA press release, “FDA modifies dosing recommendations for Erythropoiesis-Stimulating Agents — Cites increased risk of cardiovascular events when used to treat chronic kidney disease”, which included the following:

The U.S. Food and Drug Administration today recommended more conservative dosing guidelines for Erythropoiesis-Stimulating Agents (ESAs) when used to treat anemia in patients with chronic kidney disease (CKD) because of the increased risks of cardiovascular events such as stroke, thrombosis, and death….

Procrit —  (epoetin alfa) is a man-made form of a protein that helps your body produce red blood cells

Q1 2012 58 (3) $264,190 -2.13% 295 -4.22%
Q4 2011 55 (2) $269,937 3.58% 308 3.01%
Q3 2011 53 (12) $260,610 -21.61% 299 -21.32%
Q2 2011 41 (7) $332,466 7.04% 380 5.56%
Q1 2011 48 $310,606 360


Epogen —  (epoetin alfa) is a man-made form of a protein that helps your body produce red blood cells

Q1 2012 27 (7) $489,570 -24.54% 555 -17.04%
Q4 2011 20 (2) $648,794 4.67% 669 3.40%
Q3 2011 22 (2) $619,828 -13.96% 647 -18.41%
Q2 2011 20 (1) $720,376 3.32% 793 4.48%
Q1 2011 19 $697,224 759


Neulasta $4.2 2002 — used to prevent neutropenia, a lack of certain white blood cells caused by receiving chemotherapy. stimulates the bone marrow and promotes the growth of white blood cells called neutrophils

Q1 2012 13 (1) $849,971 3.33% 331 1.53%
Q4 2011 12 (2) $822,578 4.59% 326 3.49%
Q3 2011 14 (1) $786,464 -3.86% 315 -5.69%
Q2 2011 15 $818,068 4.04% 334 3.41%
Q1 2011 15 $786,288 323


Novolog $3.7 2000 —  Insulin aspart is a fast-acting form of insulin. NovoLog is used to treat type 1 (insulin-dependent) diabetes in adults and children who are at least 2 years old. It is usually given together with a long-acting insulin.

Q1 2012 57 (6) $266,305 5.67% 2,980 3.72%
Q4 2011 63 (3) $252,015 0.97% 2,873 -0.48%
Q3 2011 60 (1) $249,591 -0.96% 2,887 -2.66%
Q2 2011 61 (5) $252,010 3.16% 2,966 -0.70%
Q1 2011 66 $244,297 2,987


Erbitux $3.6 2004 — used to treat cancers of the colon and rectum. It is also used to treat head and neck cancer.

Q1 2012 99 (2) $171,513 2.30% 266 3.91%
Q4 2011 97 (7) $167,657 -0.15% 256 0.79%
Q3 2011 90 (3) $167,909 -2.48% 254 -1.93%
Q2 2011 93 (2) $172,185 -0.89% 259 -0.38%
Q1 2011 95 $173,735 260


Aranesp $3.2 2001 — Anemia, low RBC,  (darbepoetin alfa) is a man-made form of a protein that helps your body produce red blood cells. 

Q1 2012 72 (6) $231,643 -5.86% 293 -7.86%
Q4 2011 66 (15) $246,056 -6.07% 318 -3.64%
Q3 2011 51 (3) $261,967 -10.25% 330 -11.29%
Q2 2011 48 (3) $291,873 -1.03% 372 -1.33%
Q1 2011 51 $294,912 377


The article reports on the decline of worldwide sales of Aranesp drug from Thousand Oaks, California-based Amgen Inc. as of the second quarter of 2007. According to Amgen, the 10% decrease of Aranesp worldwide sales was due to the reimbursement issues related to the anemia drug and the drop of U.S. demand for drug, in which the U.S. Aranesp reported sales in the second quarter of 2007 was only $578 million from $713 million in 2006.


1/24/2011, Amgen boosts prices to offset Aranesp sales

Amgen is hiking prices to make up for the shrinking sales volume of its anemia drug Aranesp. Bloomberg reports that Amgen raised the price tag on Aranesp itself by 4.4 percent, but also marked up the white-blood-cell-boosting meds Neulasta and Neupogen by 2.9 percent.


Recombinate $2.9 1998 — Antihemophilic Factor VIII (Recombinant)

BioScience core franchises include: Hemophilia, Biotherapeutics, BioSurgery and Vaccines. BioScience products represent approximately 45 percent of Baxter’s annual sales, totaling $5.7 billion in 2010.

2007 Outlook – Sales within Baxter’s BioScience business totaled $1.2 billion, an increase of 18 percent from the same period last year. This growth was driven by record sales of ADVATE, Antihemophilic Factor (Recombinant), Plasma/Albumin Free Method (rAHF-PFM) for the treatment of hemophilia A, antibody therapy products, including GAMMAGARD LIQUID(TM) [Immune Globulin Intravenous (Human)] (IVIG) 10% Solution for the treatment of primary immunodeficiencies, specialty plasma therapeutics and biosurgery products. Medication Delivery sales increased 7 percent to $1.0 billion, with increased sales of infusion systems, intraveneous solutions and parenteral nutrition products, along with accelerated growth in the company’s drug delivery business. Renal sales increased 6 percent to $537 million reflecting accelerating gains in peritoneal dialysis patients globally.

Lucentis $2.7 2006 intraocular injection. (ranibizumab injection) is a recombinant humanized IgG1 kappa isotype monoclonal antibody fragment designed for intraocular use. Ranibizumab binds to and inhibits the biologic activity of human vascular endothelial growth factor A (VEGF-A).

Date Range Sales Rank Sales ($000) Units (000)
Q1 2012 35 (5) $409,547 -6.89% 224 -5.88%
Q4 2011 30 (2) $439,867 2.44% 238 2.59%
Q3 2011 28 (2) $429,393 1.13% 232 0.87%
Q2 2011 30 (3) $424,611 0.95% 230 1.32%
Q1 2011 33 $420,635 227



Lucentis brought in $1.7 billion for Roche last year, according to data compiled by Bloomberg.Alimera Sciences Inc. (ALIM), based in Alpharetta, Georgia, and Psivida Corp. (PSDV) also are developing a diabetic macular edema treatment known as Iluvien. The FDA has twice rejected Iluvien, most recently in November.

The FDA pooled results from two Roche clinical trials and found 39 percent of patients who used the 0.3 milligram dose were able to read three additional lines of letters on an eye chart after two years compared to 41 percent who had the same effect on the 0.5 milligram dose, according to an FDA staff report released July 24.

Genentech recommended approval of the 0.3 milligram dose in its application to the FDA since there isn’t evidence of additional benefit of the higher dose, Terence Hurley, a spokesman for the company, said in an e-mail.

Patients who received the monthly injection also were significantly more likely than those who received fake doses of the drug to achieve 20/40 vision, enough eyesight to drive.


Avonex $2.6 1996 —  Multiple Sclerosis, a form of protein called beta interferon that occurs naturally in the body. Interferons help the body fight viral infections. Avonex is used to treat patients with relapsing forms of multiple sclerosis to slow the accumulation of physical disability. This medication will not cure MS, it will only decrease the frequency of relapse symptoms.

Q1 2012 39 (1) $388,623 2.22% 130 -3.70%
Q4 2011 38 (5) $380,189 0.19% 135 -2.17%
Q3 2011 33 $379,457 -0.05% 138 -1.43%
Q2 2011 33 (4) $379,639 2.45% 140 -1.41%
Q1 2011 37 $370,570 142


Second-quarter net income surged 34 percent to $386.8 million, or $1.61 a share, from $288 million, or $1.18, a year earlier, the Weston, Massachusetts-based company said today in a statement. Earnings excluding some items of $1.82 topped by 26 cents the average of 21 analysts’ estimates (BIIB) compiled by Bloomberg. Revenue beat estimates by about $90 million.

Biogen said profit this year is expected to be more than $6.20 a share, 5 cents higher than its May 1 forecast (BIIB). The company has been increasing sales of Avonex, Rituxan and Tysabri, another MS therapy, while developing new medicines to introduce to the market.


AVONEX(R) (interferon beta-1a) revenues increased 16% year-over-year to $762 million.

Novolin $2.5 1991  —  Novolin R (insulin regular) is a short-acting form of human insulin, Diabetes, Type 1 Type 2

Date Range Sales Rank Sales ($000) Units (000)
Q1 2012 74 (2) $227,228 8.96% 2,489 13.81%
Q4 2011 76 (4) $208,552 10.19% 2,187 6.73%
Q3 2011 80 (6) $189,267 4.15% 2,049 4.92%
Q2 2011 86 (7) $181,733 3.71% 1,953 29.60%
Q1 2011 93 $175,235 1,507
Novo Nordisk launches iPhone app Posted 17th September 2010, 15:11:54
An iPhone app has been launched by Novo Nordisk in the US which lets healthcare staff check dosage guidelines for diabetes patients.

Novo Dose provides product-specific data for the company’s insulin analog agents Levemir (insulin detemir), NovoLog (insulin aspart) and NovoLog Mix (insulin aspart protamine/insulin aspart injectable).

Combined sales of the three medications increased by 24% last year, feeding a double-digit growth in Novo Nordisk sales and profits.

Novo Dose, the second diabetes app created by the industry, tells professionals when and how to dose the drugs, how to titrate and provides information on the blood glucose goals of patients.

Commenting on the new technology, Anup Kumar Sabharwal, an endocrinologist at the University of Miami Clinics’ Diabetes Research Institute, said: “This is where modern medicine is headed.”

Humalog $2.2 1996  Humalog is used to treat type 1 (insulin-dependent) diabetes in adults. Insulin lispro is a fast-acting form of insulin. It is usually given together with another long-acting insulin. It works by lowering levels of glucose in the blood. Humalog is also used together with oral (taken by mouth) medications to treat type 2 (non insulin-dependent) diabetes in adults.

Q1 2012 65 $244,587 -2.70% 2,570 -3.85%
Q4 2011 65 (2) $251,367 3.78% 2,673 2.81%
Q3 2011 63 (4) $242,208 -0.75% 2,600 -1.78%
Q2 2011 67 (5) $244,050 4.83% 2,647 1.15%
Q1 2011 72 $232,809 2,617


Pegasys $2.0 2002 — (peginterferon alfa-2a) is made from human proteins that help the body fight viral infections. Pegasys is used to treat chronic hepatitis B or C. It is often used together with another medication called ribavirin (Copegus, Rebetol, RibaPak, Ribasphere, RibaTab).

Q1 2012 90 (1) $181,693 3.92% 87 3.57%
Q4 2011 91 () $174,833 84


Rebif $1.7 2002 — (interferon beta-1a) is a protein identical to one found in the body. Interferon beta-1a is made from human proteins. Interferons help the body fight viral infections. Rebif is used to treat relapsing multiple sclerosis (MS). This medication will not cure MS, it will only decrease the frequency of relapse symptoms.

Q1 2012 62 (1) $258,088 -0.21% 540 -9.09%
Q4 2011 61 (7) $258,643 0.43% 594 -0.34%
Q3 2011 54 (5) $257,535 1.48% 596 -1.49%
Q2 2011 59 (2) $253,780 0.25% 605 -0.66%
Q1 2011 61 $253,143 609


Cerezyme $1.5 1994 —  Gaucher disease and Fabrazyme for Fabry disease.

Last year Genzyme was forced to temporarily close its manufacturing plant in Boston due to a viral contamination. The interruption lead to shortages of two key drugs: Cerezyme for Gaucher disease and Fabrazyme for Fabry disease.

That crisis sent the company’s stock price plummeting from nearly $84 in 2008 to a low earlier this year of $45.39. Sanofi’s offer to acquire the company for $18.5 billion, or $69 a share — along with a 14 percent rise in the NYSE Arca Biotech Index since late July — have helped the shares rebound.

But Genzyme is now on a mission to prove to shareholders that it is worth more than Sanofi is offering, and executives told investors on a conference call that the third quarter marks the beginning of its financial turnaround.

Third-quarter sales of Cerezyme, the company’s top drug, rose to $179.8 million from $93.6 million a year earlier, beating analysts’ average forecast of $175 million.

“In the third quarter we saw our financial recovery start to take effect, and we expect that this will accelerate during the fourth quarter as Cerezyme patients are able to return to normal dosing levels and we begin to increase shipments of Fabrazyme,” Genzyme CEO Henri Termeer said in a statement.

Cerezyme is the principal drug for Gaucher patients. In the first quarter of 2012 Genzyme (now part of Sanofi (SNY))reported Cerezyme sales of 149 million euros (approx. $194 million), up 5.8% from the same quarter of the previous year. The other supplier Shire (SHPGY) reported $72 million in Vpriv sales, up 22%. There is now a third supplier, Pfizer (PFE), teamed up with the Israeli company Protalix Biotherapeutics (PLX), whose product was approved by the FDA in May 2012. Elelyso (taliglucerase alfa) is now available in the US.
Product Cerezyme
2009 2010 2011
 Total 793 720 885
 Ann. Growth Total -9% 23%


Tysabri $1.4 2004 — Multiple Sclerosis by Elan and Biogen

Global in-market sales of TYSABRI in the second quarter of 2012 were $395 million, an increase of 2% over the second quarter of 2011. The total was comprised of $211 million in U.S. sales and $184 million in sales outside the U.S.

TYSABRI(R) (natalizumab) revenues were $280 million, in-line with the second quarter of 2011.

Elan derives its revenue almost exclusively from Tysabri and it reported total sales for the three months to June 30 of $288 million, up 6 percent on a year ago once sales from its since-divested drug delivery business are omitted.

That compared to the $299 million forecast by four analysts surveyed by Reuters and was driven by in-market sales of Tysabri that rose 2 percent year-on-year to $395 million, also shy of the $419 million expected by analysts.

Biogen, which detailed the sales numbers when it reported second quarter results on Tuesday, attributed the softer-than-expected Tysabri sales to a dispute with the Italian government over pricing.

The number of patients on Tysabri rose 4 percent to 69,100, maintaining Elan and Biogen’s 10 to 12 percent share of the MS drug market in the face of competition from Swiss drugmaker Novartis AG’s Gilenya treatment, the first multiple sclerosis pill to come on the market.

The average addition of 185 new patients per week was the highest quarterly run-rate since the fourth quarter of 2009.


NovoSeven $1.4 1999 —  Anti-fibrinolytics by Novo Nordisk —

Generic Name:   eptacog alfa
Product NovoSeven
2009 2010 2011
 Total 1,324 1,431 1,559
 Ann. Growth Total 8% 9%


Synagis $1.3 1998 — Generic Name:  palivizumab     Anti-virals by AstraZeneca

Product Synagis
2009 2010 2011
 Total 1,042 906 570
 Ann. Growth Total -13% -37%


Neupogen $1.3 1991 —  (filgrastim) is a man-made form of a protein that stimulates the growth of whiteblood cells in your body. White blood cells help your body fight against infection. Neupogen is used to treat neutropenia, a lack of certain white blood cells caused by cancer,bone marrow transplant, receiving chemotherapy, or by other conditions.

Q1 2012 70 $238,427 0.06% 170 -2.86%
Q4 2011 70 (5) $238,289 0.16% 175 10.76%
Q3 2011 65 (5) $237,915 0.69% 158 0.64%
Q2 2011 70 (4) $236,294 2.51% 157 0.64%
Q1 2011 74 $230,515 156


Betaseron $1.2 1993 — (interferon) is made from human proteins. Interferons help the body fight viral infections. Betaseron is used to treat relapsing multiple sclerosis (MS). Betaseron will not cure MS, it will only decrease the frequency of relapse symptoms.

Q1 2012 98 (1) $172,143 2.93% 67 -10.67%
Q4 2011 99 (12) $167,236 -3.76% 75 -5.06%
Q3 2011 87 (2) $173,769 -2.89% 79 -2.47%
Q2 2011 89 (4) $178,938 -2.17% 81 -7.95%
Q1 2011 85 $182,908 88


Humulin $1.1 1992 Insulin Human by Eli Lilly 

Product Humulin R
2009 2010 2011
 Total 1,022 1,089 1,249
 Ann. Growth Total 7% 15%


Kogenate FS $1.1 1993 — octocog alfa    Anti-fibrinolytics By Bayer

Product Kogenate
2009 2010 2011
 Total 1,238 1,332 1,496
 Ann. Growth Total 8% 12%



Biosimilars are defined as biological products similar, but not identical, to the reference biological products that are submitted for separate marketing approval following patent expiration of the reference biological products. As one of the ICH members, the US needs to catch up with the EU and Japan as those two countries have already issued regulatory guidelines for biosimilars.

Once Congress establishes a legal framework, FDA is expected to set up a biosimilar approval pathway which will be similar to those in the EU and Japan and harmonized under ICH. The biosimilar will need a full CMC development package plus demonstration of comparable quality attributes and comparable efficacy and safety to the innovator’s product. Table 5 provides a comparison summary between small-molecule generics and biosimilars. It will take a much bigger effort to develop a biosimilar than a generic drug. Automatic substitution between the innovator product and a biosimilar is not appropriate as a biosimilar is not a generic version of the innovator product and is approved based on comparability to the innovator product.



Read Full Post »

Biosimilars: CMC Issues and Regulatory Requirements

Reporter: Aviva Lev-Ari, PhD, RN

Updated on 6/30/2015

Biosimilars in the US: How much can we learn from Europe?


Updated on 2/10/2014

Cambridge Healthtech    Institute’s Fifth  Annual Biotherapeutics Analytical Summit
Hyatt Regency    Baltimore | Baltimore,  MD | BiotherapeuticsAnalyticalSummit.com


The    Science and  Regulation of Process Changes for Biologics (Comparability)
Thursday,  March 27, 2014 | 5:30 – 8:30 PM | More Information 


Manufacturing changes can impact on quality     attributes of biologics, and may affect efficacy and/or safety of the product.   For that reason, a thorough comparability exercise is required, to assess the   impact of the change and whether CMC data alone will suffice to support the     change. This interactive short course will consider comparability exercises     during development, as well as post-approval, addressing regulatory and     technical requirements. This should provide the attendee with the knowledge on   how to prepare a comparability package for discussion with regulatory  agencies,   towards acceptance of the proposed change to the process/product.  Attendees   will  be contacted before the event and asked about topics on which  they would   like to  focus.

Topics covered     include:

  • Ways that manufacturing changes can impact    on quality attributes
  • Features of a thorough comparability exercise
  • Critical evaluation of quality data
  • The comparability exercise during development
  • Post-approval comparability, ICH Q5E, Comparability Protocols (US) and Change Management Protocols (EU)
  • Regulatory requirements in the EU and US: guidelines, their interpretation and application
  • Discussion with Q&A

Course    Instructors:
Christopher J. Holloway, Ph.D., Group     Director, Regulatory Affairs &  CSO, ERA Consulting Group        
Kazumi Kobayashi, Ph.D., Director, Bioprocess Development, Biogen Idec,    Inc.       
Marjorie Shapiro, Ph.D., Chief, Laboratory of Molecular and Developmental  Immunology, Division of Monoclonal Antibodies,            FDA/CDER

Comparability and Developability conference program at Biotherapeutics Analytical Summit.

The third track of the Biotherapeutics Analytical    Summit focuses on the practical application of analytical characterization for    Comparability, Biosimilarity and Development purposes. It covers case studies    with a variety of products and a range of analytical technologies. We have    invited the FDA and regulatory experts to advise and to discuss regulatory    challenges being experienced by the industry. This conference also covers the    link between the process and analytical technologies for innovator products and   for biosimilars.




From: Biotherapeutics Analytical Summit Short Course <lauran@healthtech.com>
Date: Mon, 10 Feb 2014 12:40:59 -0500
To: <avivalev-ari@alum.berkeley.edu>
Subject: The Science and Regulation of Process Changes for Biologics (Comparability)

Comparability: The Final Frontier of Protein Therapies

The rapid expansion of protein therapeutics has crossed into all major disease classes (cancer, metabolic disease, inflammatory disease, infectious disease, immune disorders, etc.).  Due to the lengthy learning curve and high cost of developing these complex products, the development of therapies has been traditionally limited to highly specialized companies.  As protein therapeutics become more mainstream, these products are finding new applications in disease treatment and commercial application beyond the range of the traditional biotech companies.  Unfortunately, the expansion of medical applications for these therapies is outpacing the rate of innovation in product development and, as a result, market availability is becoming constrained by the ability to characterize and control product characteristics.

The Future Opportunity:

The key to most effectively and efficiently developing a biopharmaceutical or biologic to marketing approval is to have a clear understanding of the unique properties of proteins and to use that knowledge to design appropriate manufacturing processes, preclinical pharmacology-toxicology and clinical programs.  It is essential in dealing with complex comparability issues related to the types of manufacturing processes or changes in manufacturing to understand the scientific reasons for requiring a demonstration of comparability and the relationships between bioanalytical differences or changes in a protein and potential alterations in protein functionality in terms of specificity, potency, pharmacological activity, pharmacokinetics, toxicity and ultimately clinical safety and efficacy.  This understanding requires in depth knowledge of protein chemistry, manufacturing processes, pharmacology, immunology and toxicology that comes with extensive training and experience.  The same scientific and regulatory expertise and experience that is required for a successful demonstration of comparability is also applicable to the development of a biosimilar. This type of experience and expertise should prove invaluable as the expansion in the area of protein therapies continues and the development of biosimilars grows in the coming years.

How to Get There from Here:

Product developers can begin today to capitalize on this opportunity.   A number of technology advancements are being explored that will enhance our understanding of relationships between process, product, and clinical safety and efficacy.   One must think through how to integrate the future product comparability into early stage product development.  For new product development, these issues will include more in-depth analysis of the product structure and relationship of various structural features to function andin vivo activity, increased knowledge about the effect of process conditions on the types and mix of final product variants, and careful choice of in vitro binding and functional assays that clearly relate to the proposed mechanism of action of the product and can often also be used as a potency assays.  New technologies are being developed for these assays, and appropriate in vitro functional assays as relates to pharmacological mechanism of action can be very useful for demonstrating comparability.

A careful determination of appropriate animal models of disease for demonstration of proof-of-concept pharmacology is also important early in development. Identification of appropriate biomarkers of efficacy or safety should also be examined in these early animal models for future use in clinical development as well as demonstration of comparability.  The discovery of appropriate biomarkers can sometimes be carried over to the clinic and used in clinical trials with the appropriate validation.  Finally, as the safety database for the various classes of biopharmaceuticals and biologics expands, the understanding of safety issues associated with each of these various product classes will make it easier to more efficiently demonstrate comparability as well as to develop biosimilar products.

For developers who are trying to bridge comparability on products that lack a complete process/product history due to legacy issues or as in the case for biosimilar development, companies must think about the pharmacology of the product as relates to the proposed indication of either the previous iteration of the product or the innovator product, depending on whether this is a comparability issue or development of a biosimilar. It is the pharmacological activity of a given protein product that determines the efficacy and to a great extent the toxicity of the product.   The pharmacological activity of a product is driven by protein structure, mix of product variants, binding kinetics, dose, dosing regimen, route of administration, and final product formulation, among others.

As the development of biopharmaceuticals and biologics continues to expand, more and more information accrues on potential safety issues related to each of the various product classes, and this information will also prove quite useful to demonstration of comparability and development of biosimilars.

Biologics Consulting Group can assist developers in designing and implementing each of their comparability programs with the greatest chance of rapid regulatory approval.  Our staff has both FDA and industry experience, with a track record of success in helping academic institutions, start-ups, and established biotech and pharma companies.  Our direct knowledge and contemporary experience with all possible regulatory pathways – and every associated nuance — and can provide the requisite preclinical, clinical, quality, analytical, and manufacturing support to increase your chances for success.

Contributors: T. CarrierD. BarngroverJ. JessopV. NarbutJ. HumphriesB. FraserR. WolffN. RitterL. Winberry 



For IP and Legal aspects of Biosimilars, go to:

Biosimilars: Intellectual Property Creation and Protection by Pioneer and by Biosimilar Manufacturers


For Financial Aspects of Biosimilars, go to:

Biosimilars: Financials 2012 vs. 2008


Tr e n d s  i n  B i o / P h a r m a c e u t i c a l I n d u s t r y , 1 9 -26

Special Report on Biosimilars

About the Author: Dr. Bao-Lu has over 18 years of experience in product development, CMC regulatory, manufacturing management and quality oversight. He is currently the Director of Manufacturing and Process Development at Sangamo BioSciences. In this role, he oversees outsourced GMP production and testing of Sangamo’s gene therapy products and is responsible for the release and disposition of final drug product. He also provides CMC regulatory support and manages the in-house quality system by maintaining GMP database and implementing quality SOPs. Previously, Bao-Lu served as an Associate Director of Formulation at Xencor and Chiron and a Formulation Scientist at Amgen. Bao-Lu graduated with a BS degree from Fudan University and was selected as one of the forty chemistry students in the first year CGP Doering program. Bao-Lu earned his Ph.D. in Chemistry from University of Oregon and performed postdoctoral research in Biology at Massachusetts Institute of Technology.


CMC Issues and Regulatory Requirements for Biosimilars


Chemistry, Manufacturing and Controls (CMC), preclinical and clinical are three critical pieces in biosimilars development. Unlike a small-molecule generic drug, which is approved based on “sameness” to the innovator’s drug; a biosimilar is approved based on high similarity to the original approved biologic drug. This is because biologics are large and complex molecules. Many functional-, safety- and efficacy-related characteristics of a biologic depend on its manufacturing process. A biosimilars manufacturer won’t be able to exactly replicate the innovator’s process. The traditional abbreviated pathway for generic drug approval through the Hatch- Waxman Act of 1984 doesn’t apply for biosimilars as drugs and biologics are regulated under different laws. New laws and regulations are needed for biosimilars approval in the US. The EU has issued biosimilars guidelines based on comparative testing against the reference biologic drug (the original approved biologic). A full scale CMC development is required including expression system, culture, purification, formulation, analytics and packaging. The manufacturing process needs to be developed and optimized using state-of-the-art technologies. Minor differences in structure and impurity profiles are acceptable but should be justified. Abbreviated clinical testing is required to evaluate surrogate markers for efficacy and demonstrate no immunogenic response to the product.

We anticipate the package for a biosimilars approval in the US will be similar to that in the EU and contain a full quality dossier with a comparability program including detailed product characterization comparison and reduced preclinical and clinical requirements.

Biosimilars Become Inevitable

 Biologics developed through biotechnology constitute an essential part of the pipeline for medicines available to patients today. Biologic drugs are quite expensive and many of them are top-selling medicines (see Table 1). Since they come at extremely high prices to consumers, some patients may not be able to afford the use of biologics as the best-available treatments to their conditions. The patent protection on a large number of biologics has expired since 2001. These off-patent biologics include Neupogen, Novolin, Protropin, Activase, Epogen or Procrit, Nutropin, Humatrope, Avonex, Intron A, and Humulin. Traditionally, when a drug patent expires, a generic drug will be quickly developed and marketed. Similarly, generic version of off-patent biologic drugs (also referred to biosimilars or follow-on biologics or biogenerics) represents an extraordinary opportunity to companies that want to seize the potentially great commercial rewards in this unexploited territory. Biosimilars not only benefit the biosimilar manufacturers but also can save patients, and insurance companies, substantial cost and allow patients to gain access to more affordable biologics resulting in market expansion. The government can use biosimilars to reduce healthcare costs. Therefore, development and marketing of bosimilars are supported by both manufacturers and consumers.

Differences between Generic Drugs and Biosimilars

Enacted in 1984, the US Drug Price Competition and Patent Term Restoration Act, informally known as the “Hatch-Waxman Act of 1984” standardized US procedures for an abbreviated pathway for the approval of small-molecule generic drugs. The generic drug approval

is based on “sameness”. In comparison to the innovator’s drug, a generic drug is a product that has the same active ingredient, identical in dose, strength, route of administration, safety, efficacy, and intended use. For approval, the generic companies can go through the Abbreviated

New Drug Application (ANDA) process with reduced requirement in comparison to approval for a new drug entity. The generic drugs need to show bioequivalence to the innovator drugs typically based on pharmacokinetic parameters such as the rate of absorption or bioavailability in 24 to 36 healthy volunteers. No large clinical trials for safety and efficacy are required. The generic companies can rely on the FDA’s previous findings of safety and effectiveness of the innovator’s drugs.

However, the abbreviated pathway for generic drugs legally doesn’t apply to biologics as small-molecule drugs and biologics are regulated under different laws and approved through different pathways in the US (Table 2). Small-molecule drugs are regulated under the Food, Drug and Cosmetic Act (FD&C) and require submission of a New Drug Application (NDA) to FDA for drug review and approval. Biologics are regulated under the Public Health Service Act (PHS) and require submission of a Biologic License Application (BLA) to FDA for review and approval. The Hatch-Waxman Act of 1984 doesn’t apply for biosimilars. New laws are needed to establish a pathway for biosimilar approval.

There are some crucial differences between biologics and small-molecule drugs (Table 3). Small-molecule drugs are made from chemical synthesis. They are not sensitive to process changes. The final product of a small-molecule drug can be fully characterized. The developmentand production of generic drugs are relatively straightforward. Biologics are made from living organisms so that its functional-, efficacy- and safety-related properties depend on its manufacturing and processing conditions. They are sensitive to process changes. Even minor modifications of the manufacturing process can cause variations in important properties of a biological product. Thus it is believed that a biologic product is defined by its manufacturing process. Biologics are 100- or 1,000-fold larger than small-molecule drugs, possess sophisticated three-dimensional structures, and contain mixtures of protein isoforms. A biological product is a heterogeneous mixture and the current analytical methods cannot characterize these complex molecules sufficiently to confirm structural equivalence with the reference biologics.

Laws and Regulatory Pathways for Drug Approval in the US

Law/Application           Small-molecule            Drug Biologics                     

Law                     Food, Drug and Cosmetic Act (FD&C)             Public Health Service Act (PHS)

Drug application                 New Drug Application (NDA)   Biologic License Application (BLA)

Generic application   Abbreviated New Drug   Application(ANDA)   No pathway yet

 Immunogenicity Poses a Concern

One of the major complications that biologics can produce is immunogenicity as therapeutic proteins are inherently immunogenic [2]. Immunogenicity is related to biologics structure and formulation and is dependent on dose, route of administration and frequency of administration.

Clinical implications of immunogenicity are not always predictable. Formation of antibodies can result in harmless clinical effect or produce significant adverse events or severe disease. Examples are provided below. The Eprex (Erythropoietin, EPO) has been marketed by Johnson & Johnson (J&J) in the European Union (EU) countries for 10 years with no noticeable

Differences between small-molecule drugs and biologics

Product characteristics

Small-molecule generics Small, simple molecule

(Molecular weight: 100-1,000 Da)

Biosimilars   Large, complex molecules, Higher order structures, Post-translational, modifications

(Molecular weight: 15,000-150,000 Da)


Small-molecule generics Produced by chemical synthesis

Biosimilars  Produced in living organisms

Analytical testing

Small-molecule  Well-defined chemical structure, all its various components in the finished drug can be determined

Biosimilars  Heterogeneous mixture, difficult to characterize, some of the components of a finished biologic may be unknown

Process dependence

Small-molecule   Not sensitive to manufacturing process changes. The finished product can be analyzed to establish the sameness.

Biosimilars   Sensitive to minor changes in manufacturing process. The product is defined by the process

Identity and purity

Small-molecule Often meeting pharmacopeia or other standards of identity (e.g., minimums for purity and potency)

Biosimilars   Most have no pharmacopeia monographs

immunogenicity issues prior to 1998. When J&J made a change in the Eprex formulation by replacing human serum albumin (HAS) with polysobate 80 and glycine in response to the

request from European health authorities, some patients developed pure red-cell aplasia (PRCA), a severe form of anemia. Eprex induced antibodies neutralize all the exogenous rHuEPO and cross-react with endogenous erythropoietic proteins. As a result, serum EPO is undetectable

and erythropoiesis becomes ineffective. Upon investigation, J&J found that polysorbate 80 might have caused uncoated rubber stoppers in single-use Eprex syringes to leach plasticizers, which stimulated an immune response that resulted in PRCA. Replacing with Teflon coated stoppers resulted in 90% decrease in PRCA by 2003 [3,4]. The effect of neutralizing antibodies has not always resulted in serious clinical consequences. Three interferon beta products, Betaseron, Rebif and Avonex, are marketed by three different companies. These products induce neutralizing antibodies in multiple sclerosis patients from 5 to 50% after one year treatment. Although these antibodies might be associated with loss of efficacy of treatment resulting in some patients to withdraw from the treatment, it seems no other severe adverse effects were detected [5,6].

Regulatory Landscape

The US, the EU and Japan are the three cornerstonemembers of the International Conference on Harmonization (ICH), which intends to harmonize the regulatory requirements for drug or biologic approval in these three regions. With the other two members, the EU and Japan, already have established biosimilar approval procedures (see below), the US lags behind in the biosimilar race. There are no formal approval pathways for biosimilars in the US. Congress needs to establish a legal framework in order for FDA to develop guidelines. Legislation has been under discussion in Congress since 2007. The legislative debate is centered on patient safety and preserving incentives to innovate with introduction of biosimilars. Two bills introduced in March 2009 deserve attentions [7,8]. The Waxman bill (H.R. 1427) proposes 5 years of market exclusivity to the innovator companies and requires no clinical trials for biosimilar development. The Eshoo bill (H.R. 1548) proposes 12 years of market exclusivity to the innovator companies and requires clinical trials for biosimilar development. Obama administration appears to favor a 7-year market exclusivity [9]. Once a legal framework is established for biosimilars, the FDA will likely take a conservative approach using the comparability as an approval principle. Clinical proof of efficacy and safety will be required, probably in reduced scale.

In the EU, the European Medicines Agency (EMEA) issued regulatory guidelines for approving biosimilars in 2005 (Figure 1) [10-16]. These include two general guidelines for quality issues [11] and non-clinical and clinical issues [12] and four class-specific annexes for specific data requirements for Granulocyte-Colony Stimulating factor (G-CSF) [13], Insulin [14], Growth hormone [15] and Erythropoietin [16]. In addition, a concept paper on interferon alpha [17] is also available. So far, there are eleven biosimilar products which received market authorization in the EU and they are biosimilar versions of human growth hormone, Epoetin and filgrastim. It is estimated six to eight years on average for a biosimilar to be developed [18].

The EMEA treats a biosimilar medicine as a medicine which is similar to a biological medicine that has already been authorized (the “biological reference medicine”) in the EU, The active substance of a biosimilar medicine is similar to the one of the biological reference medicine.

A biosimilar and the biological reference medicine are used in general at the same dose to treat the same disease. A biosimilar and the biological reference medicine are not automatically interchangeable because biosimilar and biological reference medicine are only similar but not identical. A physician or a qualified healthcare professional should make the decision to treat a patient with a reference or a biosimilar medicine. Since the biosimilar may contain different inactive ingredients, the name, appearance and packaging of a biosimilar medicine differ to those of the biological reference medicine. In addition, a pharmacovigilance plan must be in place for post-marketing safety monitoring.

Japan’s Ministry of Health, Labor and Welfare (MHLW) issued guidelines for follow-on proteins or biosimilars approval in March 2009. The first biosimilar, Sandoz’ growth hormone Somatropin, was approved in June 2009. The MHLW’s guidelines consider biosimilars drugs which are equivalent and homogeneous to the original biopharmaceuticals in terms of quality, efficacy and safety. Biosimilars are also requested to be developed with updated technologies and knowledge. Biosimilars need to demonstrate enough similarity to guarantee the safety and efficacy instead of absolute identity to the original biologics. Biosimilars’ regulatory approval applications will be categorized separately from conventional generic drugs. In general, the applications should be submitted, as the new drug applications, with data from clinical trials, manufacturing methods, long-term stability and information on overseas use. The MHLW will assess the data on absorption, distribution, metabolism and excretion (ADME) on a case-by-case basis. The applications do not need to provide data on accessory pharmacology, safety pharmacology and genotoxicity.

Biosmilars are already thriving in Eastern Europe and Asia, where regulatory and intellectual property (IP) standards for biosimilars are more liberal. Biosimilars developed in these regions are primarily sold domestically. These markets are considered less controlled. The quality of the biosimilars may not be in full compliance with ICH guidelines although they are often developed through comparative quality testing and clinical trials against the biologics which are already approved in Western countries

CMC Development

The CMC requirements for biosimilars in the EU are those described in the ICH Common Technical Document (CTD) Quality Module 3 with supplemental information demonstrating comparability or similarity on quality attributes to the reference medicine product.

Since the US is a member of ICH and encourages submission using CTD format, once the legal framework for approving biosimilars is established in the US, the CMC development will be similar to those in the EU.

Biosimilar manufacturers will have no access to the manufacturing process and product specifications of the innovator’s products because these are proprietary knowledges. To develop a biosimilar, a biosimilar manufacturer will need to first identify a marketed biologic product to serve as the reference biologic product. Then a detailed characterization of the reference biologic product will be performed. The information obtained from the characterization of the reference biologic product will be utilized to direct the process development of the biosimilar product and comparative testing to demonstrate bioequivalence between the biosimilar product and the reference biologic product. A biosimilar will be manufactured from a completely new process, which may be based on different host/vector system with different process steps, facilities and equipment.

A flow chart for a typical work flow from production to drug use is shown in Figure 2. The CMC development starts with establishment of the expression system. A cell-line will be selected among bacterial, yeast and mammalian host strains and then the correct DNA sequence will be inserted. Elaborate cell-screening and selection methods are then used to establish a master cell bank. Extensive characterization on the master cell bank needs to be carried out to provide microbiological purity or sterility and identity [19].

Bulk protein production involves developing robust and scalable fermentation and purification processes. The goals for fermentation are to increase the expression level and efficiency without compromising the correct amino acid sequence and post translational modification. Achieving high expression requires optimizing culture medium and growth conditions, and efficient extraction and recovery procedures. Correct amino acid sequence and post translati0nal modification will need to be verified.

Cell Bank



Drug Substance



Drug Product



Typical flow chart for a biologics from production to drug use, above

Solubilization and refolding of insoluble proteins are sometimes necessary for proteins which have tendency to aggregate under the processing condition. Differences in the cell bank and production processes may create impurities that are different from the innovator’s product. The purification process needs to remove impurities such as host-cell proteins, DNA, medium constituents, viruses and metabolic by-products as much as possible. It is important for biosimilar manufacturers to accept appropriate yield losses to achieve high purity, because any increase in yield at the expense of purity is unacceptable and can have clinical consequences.

The final product is produced by going through formulation, sterile filtration and fill/finish into the final containers. Selection of formulation components starts from basic buffer species for proper pH control and salt for isotonicity adjustment. Surfactants may be needed to prevent proteins from being absorbed onto container surface or water-air interface or other hydrophobic surfaces. Stabilizers are required to inhibit aggregation, oxidation, deamidation and other degradations. The container and closure system can be glass vials, rubber stoppers and aluminum seals or pre-filled syringes or IV bags. The container and closure integrity needs to be verified by sterility or dyeleak test.

Biologics are not pure substances. They are heterogeneous mixtures. Each batch of a biologic product for clinical or commercial use needs to be produced in compliance with current Good Manufacturing Practice (cGMP) and is typically tested by a panel of assays to ensure the product meets pre-defined specifications for quality, purity, potency, strength, identity and safety. The product purity is often measured by multiple assays, which measure different product related variants (biologically active) or product related impurities (biologically inactive). Biologics are parenteral drugs and filled into the final containers through the aseptic process so that microbiological control is critical. It is advisable to set up product specifications for a biosimilar within the variation of the reference biologic product. Product characterization can be performed on selected batches for primary sequence, high order structures, isoform profiles, heterogeneity, product variants and impurities and process impurity profiles. Physicochemical characterization tests include IEF, CE, HIC, LCMS, carbohydrate analysis, N & C terminal sequencing, amino acid analysis, analytical ultracentrifugation, CD and DSC [20,21]. Biologics are highly sensitive to environmental influences during storage, shipment and handling. Temperature excursion, movement, and exposure to UV light can lead to protein degradation. Product expiry needs to be based on the real time stability data. Stability program should also include accelerated or stress studies to gain insight of the degradation profiles. In-use stability studies are carried out to verify shipping conditions or handling procedures cause no detrimental effect to the drug product.

 Comparability Demonstration

 A comparability exercise based on the ICH guideline [22] needs to be performed to demonstrate that the biosimilar product and the reference biologic product have similar profiles with respect to product quality, safety, and efficacy. This is accomplished by comparative testing of the biosimilar product and the reference biologic product to demonstrate they have comparable molecular structure, in vitro and in vivo biological activities, pre-clinical safety and pharmacokinetics, and safety and efficacy in human patients. Comparison of quality attributes between the biosimilar and the reference biologic product employs physicochemical

Product release assays for biologics

Type                         Assays

Quality              Appearance, particulates, pH, osmolality

Purity                 SDS-PAGE, SEC-HPLC, IEX-HPLC, RP-HPLC

Potency             In vitro or in vivo bioactivity assays

Strength             Protein concentration by A280

Identity               Western blot, peptide mapping, isoelectric focusing

Safety                  Endotoxin, sterility, residual DNA, host cell proteins

and biological characterization. Comparability on physical properties, amino acid sequence, high order structures, post-translationally modified forms are evaluated by physicochemical tests. In vitro receptor-binding or cell-based (binding) assays or even the in vivo potency studies in animals need to be performed to demonstrate comparable activity despite they are often imprecise. Levels of product related impurities (aggregates, oxidized forms, deamidated forms) and process related impurities and contaminants (host cell proteins, residual genomic DNA, reagents, downstream impurities) need to be assessed and quantified. Stability profiles of the biosimilar product and the reference biologic product also need to be studies by placing the products under stressed conditions. The rate of degradation and degradation profiles (oxidation, deamidation, aggregation and other degradation reactions) will be compared. If unknown degradation species are detected, they need to be studied to determine if they affect safety and efficacy. If differences on product purities and stability profiles are present between the biosimilar product and the reference biologic product, these differences need to be justified using scientific knowledge or preclinical or clinical studies. Changes in the impurity profile should be justified as well.

The demonstration of comparability in quality attributes does not necessarily mean that the biosimilars and the reference biologics are identical, but that they are highly similar. In many cases, the relationship between specific quality attributes and safety and efficacy has not been fully established. For example, physicochemical characterization cannot easily predict immunogenicity and slight changes in manufacturing processes or product composition can give rise to unpredicted changes in safety and efficacy. Changes in bioavailability, pharmacokinetics, bioactivity bioactivity, and immunogenicity are the main risks associated with the manufacturing of biosimilars. In vivo studies should be designed to measure the pharmacokinetics and pharmacodynamics relevant to clinical studies. Such in vivo studies should be designed to detect response differences between the biosimilar and the reference biologic not just responses per se. In vivo studies of the biosimilar’s safety in animals may be used to research any concerns into the safety of the biosimilar in human patients. Although extensive clinical testing is not necessary for biosimilars, some degree of clinical testing is needed to establish therapeutic comparability on efficacy and safety between the biosimilar and the reference biologic product [23,24]. This includes using surrogate markers of specific biologic activity as endpoints for demonstrating efficacy, and showing that patients didn’t develop immunogenic responses to the product. In general, the approval of biosimilars will be based on the demonstration of comparable efficacy and safety to an innovator reference product in a relevant patient population. Clinical data requirement for each individual product will be different and will be determined on a case-by-case basis.

Small-molecule Generics versus Biosimilars


  • Approval based on “sameness”


  • Approval based on “high similarity”


  • Replicate the innovator’s process and product and perform a bioavailability study demonstrating similar pharmacokinetic properties


  • Full CMC development with comparative testing, conduct substantial clinical trials for efficacy and safety including immunogenicity


  • Abbreviated registration procedures in Europe and US


  • Regulatory pathway is defined in EU on “Comparability” status, no pathway yet in US under BLA


  • Therapeutically equivalent, thus interchangeable


  • Lack of automatic substitutability


  • $1 to $5 million to develop


  • $100-$200 million to develop


  • Brand-to-generic competition


  • Brand-to-Brand competition


The patent provisions of the Biosimilar Act, 2009 establish demanding and time-sensitive disclosure requirements. ObamaCare upheld by the Supreme Court is a victory for future development of pathways for biosimilar regulatory approvaland eventually biosimilar generic drugs.

Biosimilars are defined as biological products similar, but not identical, to the reference biological products that are submitted for separate marketing approval following patent expiration of the reference biological products. As one of the ICH members, the US needs to catch up with the EU and Japan as those two countries have already issued regulatory guidelines for biosimilars. 2009 and 2012 represent milestones in the regulatory provisions for biosimilars in the US.

Once Congress establishes a legal framework, FDA is expected to set up a biosimilar approval pathway which will be similar to those in the EU and Japan and harmonized under ICH. The biosimilar will need a full CMC development package plus demonstration of comparable quality attributes and comparable efficacy and safety to the innovator’s product. Table 5 provides a comparison summary between small-molecule generics and biosimilars. It will take a much bigger effort to develop a biosimilar than a generic drug. Automatic substitution between the innovator product and a biosimilar is not appropriate as a biosimilar is not a generic version of the innovator product and is approved based on comparability to the innovator product.


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