Optimizing the design and geometry of T cell engaging bispecific antibodies targeting CEA in colorectal cancer.

Metastatic colorectal cancer (mCRC) remains a leading cause of cancer-related deaths, with a 5-year survival rate of only 15%. T cell engaging bispecific antibodies (TCBs) represent a class of biopharmaceuticals that redirect cytotoxic T cells towards tumor cells, thereby turning immunologically "cold" tumors "hot." The carcinoembryonic antigen (CEA) is an attractive tumor-associated antigen (TAA) that is overexpressed in over 98% of CRC patients. In this study, we report the comparison of four different TCB formats employing the antibodies F4 (targeting human CEA) and 2C11 (targeting mouse CD3ε). These formats include both antibody fragment- and IgG-based constructs, with either one or two binding specificities of the respective antibodies. The 2+1 arrangement, using an anti-CEA single-chain diabody (scDbCEA) fused to an anti-CD3 single-chain variable fragment (scFvCD3), emerged as the most potent design, showing tumor killing at subnanomolar concentrations across three different CEA+ cell lines. The in vitro activity was three times greater in C57BL/6 mouse colon adenocarcinoma cells (MC38) expressing high levels of CEA compared to those expressing low levels, highlighting the impact of CEA antigen density in this assay. The optimal TCB candidate was tested in two different immunocompetent mouse models of colorectal cancer and showed tumor growth retardation. Ex vivo analysis of tumor infiltrates showed an increase in CD4+ and CD8+ T cells upon TCB treatment. This study suggests that bivalent tumor targeting, monovalent T cell targeting, and a short spatial separation are promising characteristics for CEA targeting TCBs.

Adapting Neutralizing Antibodies to Viral Variants by Structure-Guided Affinity Maturation Using Phage Display Technology.

Neutralizing monoclonal antibodies have achieved great efficacy and safety for the treatment of numerous infectious diseases. However, their neutralization potency is often rapidly lost when the target antigen mutates. Instead of isolating new antibodies each time a pathogen variant arises, it can be attractive to adapt existing antibodies, making them active against the new variant. Potential benefits of this approach include reduced development time, cost, and regulatory burden. Here a methodology is described to rapidly evolve neutralizing antibodies of proven activity, improving their function against new pathogen variants without losing efficacy against previous ones. The reported procedure is based on structure-guided affinity maturation using combinatorial mutagenesis and phage display technology. Its use against the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is demonstrated, but it is suitable for any other pathogen. As proof of concept, the method is applied to CoV-X2, a human bispecific antibody that binds with high affinity to the early SARS-CoV-2 variants but lost neutralization potency against Delta. Antibodies emerging from the affinity maturation selection exhibit significantly improved neutralization potency against Delta and no loss of efficacy against the other viral sequences tested. These results illustrate the potential application of structure-guided affinity maturation in facilitating the rapid adaptation of neutralizing antibodies to pathogen variants.

Generation of a novel fully human non-superagonistic anti-CD28 antibody with efficient and safe T-cell co-stimulation properties.

Antibody-based therapeutics represent an important class of biopharmaceuticals in cancer immunotherapy. CD3 bispecific T-cell engagers activate cytotoxic T-cells and have shown remarkable clinical outcomes against several hematological malignancies. The absence of a costimulatory signal through CD28 typically leads to insufficient T-cell activation and early exhaustion. The combination of CD3 and CD28 targeting products offers an attractive strategy to boost T-cell activity. However, the development of CD28-targeting therapies ceased after TeGenero's Phase 1 trial in 2006 evaluating a superagonistic anti-CD28 antibody (TGN1412) resulted in severe life-threatening side effects. Here, we describe the generation of a novel fully human anti-CD28 antibody termed "E1P2" using phage display technology. E1P2 bound to human and mouse CD28 as shown by flow cytometry on primary human and mouse T-cells. Epitope mapping revealed a conformational binding epitope for E1P2 close to the apex of CD28, similar to its natural ligand and unlike the lateral epitope of TGN1412. E1P2, in contrast to TGN1412, showed no signs of in vitro superagonistic properties on human peripheral blood mononuclear cells (PBMCs) using different healthy donors. Importantly, an in vivo safety study in humanized NSG mice using E1P2, in direct comparison and contrast to TGN1412, did not cause cytokine release syndrome. In an in vitro activity assay using human PBMCs, the combination of E1P2 with CD3 bispecific antibodies enhanced tumor cell killing and T-cell proliferation. Collectively, these data demonstrate the therapeutic potential of E1P2 to improve the activity of T-cell receptor/CD3 activating constructs in targeted immunotherapeutic approaches against cancer or infectious diseases.

Generation and in vivo characterization of a novel high-affinity human antibody targeting carcinoembryonic antigen.

There are no effective treatment options for most patients with metastatic colorectal cancer (mCRC). mCRC remains a leading cause of tumor-related death, with a five-year survival rate of only 15%, highlighting the urgent need for novel pharmacological products. Current standard drugs are based on cytotoxic chemotherapy, VEGF inhibitors, EGFR antibodies, and multikinase inhibitors. The antibody-based delivery of pro-inflammatory cytokines provides a promising and differentiated strategy to improve the treatment outcome for mCRC patients. Here, we describe the generation of a novel fully human monoclonal antibody (termed F4) targeting the carcinoembryonic antigen (CEA), a tumor-associated antigen overexpressed in colorectal cancer and other malignancies. The F4 antibody was selected by antibody phage display technology after two rounds of affinity maturation. F4 in single-chain variable fragment format bound to CEA in surface plasmon resonance with an affinity of 7.7 nM. Flow cytometry and immunofluorescence on human cancer specimens confirmed binding to CEA-expressing cells. F4 selectively accumulated in CEA-positive tumors, as evidenced by two orthogonal in vivo biodistribution studies. Encouraged by these results, we genetically fused murine interleukin (IL) 12 to F4 in the single-chain diabody format. F4-IL12 exhibited potent antitumor activity in two murine models of colon cancer. Treatment with F4-IL12 led to an increased density of tumor-infiltrating lymphocytes and an upregulation of interferon γ expression by tumor-homing lymphocytes. These data suggest that the F4 antibody is an attractive delivery vehicle for targeted cancer therapy.

Selection of a PD-1 blocking antibody from a novel fully human phage display library.

Programmed cell death protein 1 (PD-1) is an immunoregulatory target which is recognized by different monoclonal antibodies, approved for the therapy of multiple types of cancer. Different anti-PD-1 antibodies display different therapeutic properties and there is a pharmaceutical interest to generate and characterize novel anti-PD-1 antibodies. We screened multiple human antibody phage display libraries to target novel epitopes on the PD-1 surface and we discovered a unique and previously undescribed binding specificity (termed D12) from a new antibody library (termed AMG). The library featured antibody fragments in single-chain fragment variable (scFv) format, based on the IGHV3-23*03 (VH ) and IGKV1-39*01 (Vκ) genes. The D12 antibody was characterized by surface plasmon resonance (SPR), cross-reacted with the Cynomolgus monkey antigen and bound to primary human T cells, as shown by flow cytometry. The antibody blocked the PD-1/PD-L1 interaction in vitro with an EC50 value which was comparable to the one of nivolumab, a clinically approved antibody. The fine details of the interaction between D12 and PD-1 were elucidated by x-ray crystallography of the complex at a 3.5 Å resolution, revealing an unprecedented conformational change at the N-terminus of PD-1 following D12 binding, as well as partial overlap with the binding site for the cognate PD-L1 and PD-L2 ligands which prevents their binding. The results of the study suggest that the expansion of antibody library repertoires may facilitate the discovery of novel binding specificities with unique properties that hold promises for the modulation of PD-1 activity in vitro and in vivo.

A novel human monoclonal antibody specific to the A33 glycoprotein recognizes colorectal cancer and inhibits metastasis.

Colorectal cancer represents the second most common cause of cancer-related death. The human A33 transmembrane glycoprotein is a validated tumor-associated antigen, expressed in 95% of primary and metastatic colorectal cancers. Using phage display technology, we generated a human monoclonal antibody (termed A2) specific to human A33 and we compared its epitope and performance to those of previously described clinical-stage anti-human A33 antibodies. All antibodies recognized a similar immunodominant epitope, located in the V-domain of A33, as revealed by SPOT analysis. The A2 antibody homogenously stained samples of poorly, moderately, and well differentiated colon adenocarcinomas. All antibodies also exhibited an intense staining of healthy human colon sections. The A2 antibody, reformatted in murine IgG2a format, preferentially localized to A33-transfected CT26 murine colon adenocarcinomas in immunocompetent mice with a homogenous distribution within the tumor mass, while other antibodies exhibited a patchy uptake in neoplastic lesions. A2 efficiently induced killing of A33-expressing cells through antibody-dependent cell-mediated cytotoxicity in vitro and was able to inhibit the growth of A33-positive murine CT26 and C51 lung metastases in vivo. Anti-A33 antibodies may thus represent useful vehicles for the selective delivery of bioactive payloads to colorectal cancer, or may be used in IgG format in a setting of minimal residual disease.

Targeted Delivery of TNF Potentiates the Antibody-Dependent Cell-Mediated Cytotoxicity of an Anti-Melanoma Immunoglobulin.

The recombinant murine IgG2a antibody TA99, directed against a melanoma antigen, was used to study combination modalities that potentiate antibody-dependent cell cytotoxicity. As previously reported, IgG2a(TA99) was extremely efficacious in preventing the growth of B16 lung metastases. However, the same antibody mediated only minimal tumor growth retardation when used to treat established neoplastic masses. The therapeutic activity of IgG2a(TA99) could be substantially enhanced by co-administration with an antibody-cytokine fusion (TA99-murine tumor necrosis factor [mTNF]), consisting of the TA99 antibody in single-chain variable fragment format fused to murine TNF. This fusion protein efficiently killed endothelial cells in vitro and displayed only minimal activity against B16 melanoma cells. In vivo, TA99-mTNF boosted the influx of natural killer cells and macrophages into B16 melanoma lesions. Therapy studies with two different administration schedules showed that the combination of TA99-mTNF and IgG2a(TA99) was superior to the individual products used as single agents. The combination treatment converted most of the tumor mass into a necrotic lesion, but a vital tumor rim eventually regrew, even when dacarbazine was included in the therapeutic regimen. The treatment modality described in this article may be applicable to the treatment of melanoma patients, given the specificity of the gp75 antigen and its conservation across species.