EGFR and HER2, both members of the epidermal growth factor receptor family (ErbB), are known to be co-expressed on various forms of cancer, such as breast, colorectal and prostate cancer, and co-expression of the two receptors is associated with poor patient prognosis. The simultaneous targeting of EGFR and HER2 has been suggested as a way to increase the selectivity and efficiency of a targeting agent in molecular imaging and therapeutic applications. Here, we report on the generation of bispecific EGFR × HER2-binding Affibody molecules. By combining the gene sequences of monomeric EGFR and HER2 binding Affibody molecules separated by a spacer sequence, six bispecific Affibody variants were constructed and evaluated, demonstrating binding to EGFR and HER2 separately as well as simultaneously. The confirmed bispecific targeting suggests that EGFR × HER2-binding Affibody molecules may become a useful tool for diagnostic and therapeutic targeting of tumors co-expressing EGFR and HER2.
Bispecific biologics, especially those that redirect T cells, can be engineered to be far more potent than traditional antibodies or antibody drug conjugates, which has spurred the development of at least 35 distinct bispecific protein formats. Increased potency, however, while obviously highly beneficial, comes at the price of increased on-target toxicity against normal tissues that also express the target antigen, which effectively reduces the already small number of known antigens that can be safety targeted, and thereby reduces the number of druggable indications and increases commercial competition.
In contrast, however, T-cell receptors (TCRs) target HLA-peptides, a previously unexplored class of molecular target. By binding HLA-peptide antigens, TCRs have the distinct advantage of being able to target any disease-associated protein, even if that protein is intracellular and not accessible to other biological therapeutics. This unique targeting capability provides the opportunity to target a wealth of well-characterized, highly disease-specific targets such as the cancer/testis family of antigens, which can be safety targeted with highly potent biologics without the problem of on-target/off tumor toxicity.
Immunocore has developed a platform of TCR-based bispecifics called ImmTACs (Immune mobilising mTCR Against Cancer) that combine a soluble, affinity enhanced TCR-based targeting system with an anti-CD3 single chain variable fragment (scFv)-based T-cell redirection effector function. ImmTACs redirect polyclonal T cells to recognize and kill cancer cells presenting as few as 10 epitopes per cell, they display pM potency, are easily manufactured in E.coli and have a clear regulatory pathway in place with both the FDA and MHRA. Immunocore's most advanced ImmTAC, IMCgp100 for the treatment of melanoma, is currently in clinical trials in both the UK and US; preliminary data suggests the drug is well-tolerated and capable of redirecting T cells within a solid tumor environment.
Bispecific antibodies based on full-length antibody structures are more optimal than fragment-based formats because they benefit from the favorable properties of the Fc region. However, the homodimeric nature of Fc effectively imposes bivalent binding on all current full-length bispecific antibodies, an attribute that can result in nonspecific activation of cross-linked receptors. We engineered a novel bispecific format, referred to as mAb-Fv, that utilizes a heterodimeric Fc region to enable a single additional variable region to be built monomerically onto an antibody. Our new bispecific format enables the simultaneous bivalent and monovalent co-engagement of distinct target antigens in a full-length context. mAb-Fv constructs co-targeting CD16 and CD3 were expressed and purified as heterodimeric species, bound selectively to their co-target antigens, maintained favorable properties of the Fc region and mediated potent cytotoxic activity by NK cells and T cells, respectively. The capacity to co-engage distinct target antigens simultaneously with different valencies is an improved feature for bispecific antibodies with promising therapeutic implications.
IL-4 and IL-13 are implicated as mediators of idiopathic pulmonary fibrosis (IPF), a disease of unknown etiology that has extremely limited therapeutic options and a mean survival time of only 3 y. Since the simultaneous blockade of IL4 and IL13 has a synergistic impact on their shared pathogenic activities compared with inhibiting either IL4 or IL13 alone, we developed the bispecific antibody SAR156597 by engineering Fv domains of humanized anti-IL4 and anti-IL13 antibodies into a bispecific format.
The format used for the generation of SAR156597 was an IgG variant. In this format, an IgG molecule (here the anti-IL4 antibody) is elongated at its N-terminus on the corresponding heavy and light chains by an additional variable domain of a second antibody (here the anti-IL13 antibody). Thus, the resulting IgG molecule is a heterotetramer composed of two heavy and two light chains, with the variable domains oriented in a tandem configuration. Therefore, we called our format TBTI (tetravalent bispecific tandem Ig); the TBTI format is similar to the DVD-Ig format developed independently at Abbott. Construction of the TBTI molecule using the humanized versions of the parental mouse monoclonal antibodies showed unexpected position effects that could be solved by re-engineering the linkers between the variable domains.
SAR156597 was shown to bind IL4 and IL13 (human and cynomolgus monkey) antigens in vitro with pM affinities and to inhibit IL4 and IL13 activities in several cell based assays at low nM range. SAR156597 targets both IL-4 and IL-13 simultaneously, suppresses IL-4/IL-13-induced human fibroblast activation in vitro, and thereby promises to be an innovative treatment for IPF and other fibrotic indications with the same underlying mechanism (e.g., scleroderma). A Phase 1 clinical study of SAR156597 is currently ongoing.
Affibody® molecules are targeted biologics based on a small (∼6.5 kDa) engineered three helical bundle protein. The origin and nature of the Affibody® molecule makes it ideal for creating multivalent or multispecific formats with high specificity and tunable affinity. Simultaneous bispecific targeting of the epidermal growth factor receptors EGFR and HER2 has been demonstrated in vitro using both mono and divalent Affibody® molecules. For in vivo therapeutic use, the kinetics may be extended up to several weeks by applying Affibody's Albumod™ technology. The core of the Albumod™ technology is the Albumin Binding Domain (ABD), a 5 kDa protein engineered to bind human serum albumin (HSA) with an exceptionally high affinity. Improved pharmacokinetic and pharmacodynamic properties of ABD-fusion proteins have been demonstrated in vivo. A HER2-targeting monomeric or dimeric Affibody® molecule in fusion with ABD, either at the amino- or C-terminal end, showed a favorable distribution profile and specific tumor growth inhibition when administered to xenografted mice. Furthermore, fusion of human granulocyte colony stimulating factor (G-CSF) to ABD prolonged the half-life of G-CSF and increased granulocyte counts in rodents. The Albumod™ technology has been optimized for human use, through an extensive mutational program aimed at reducing potential T-cell and B-cell epitopes. The successful deimmunization was confirmed in a CD4+ T-cell proliferation assay performed by Algonomics/Lonza. The lead ABD variant is in development for clinical use.
Fynomers represent a new class of binding molecules based on the Fyn SH3 domain. We will show the broad applicability of our large phage display library (comprising more than 8 × 1010 clones). In particular, we will demonstrate a novel Fynomer format comprising a highly potent bispecific inhibitor of human IL-17A and human TNFα—two cytokines which are known to play a significant role in chronic inflammatory diseases such as psoriasis and rheumatoid arthritis. The bispecific molecule COVA301 consists of the high affinity anti-IL-17A Fynomer 2C1, which has been genetically fused to the C-terminus of the heavy chain of a fully human anti-TNFα antibody.
COVA301 exhibits excellent biophysical properties: it stays monomeric and shows no signs of aggregation even after four months of storage at +4°C, −20°C or −80°C in PBS, or after repeated freeze-thaw cycles. Moreover, COVA301 was found to be stable after 5 d of incubation in human serum at +37°C. COVA301 binds to human neonatal Fc-receptor (FcRn) with the same affinity as the unmodified anti-TNFα antibody. Importantly, COVA301 simultaneously neutralized in vitro both IL-17A and TNFα with an apparent IC50 value of 30 pM. Based on these encouraging results we conclude that COVA301 has highly promising properties with a great potential for further preclinical and clinical development.