Common Light Chain Research Articles

Discovery of a common light chain bispecific antibody targeting PD-1 and PD-L1 by Hybridoma-to-phage-to-yeast (H2PtY) platform

Abstract Background Therapeutic antibody drugs targeting the PD-1 pathway are generally characterized by relatively low response rates and susceptibility to drug resistance during clinical application. Therefore, there is an urgent need for alternative therapeutic strategies to increase the immune response rate. Bispecific antibodies co-targeting PD-1 and PD-L1 may have greater potential to improve the efficacy of the immune checkpoint pathway. Method In this study, we developed a potent humanized common light chain IgG shape bispecific antibody, named JMB2005, based on Hybridoma-to-Phage-to-Yeast platform. The platform allowed us to discover common light chain bispecific antibody from traditional mice for any pair of given targets. Results JMB2005 exhibited favorable developability, good manufacturing property, and satisfactory efficacy, which could be given via subcutaneous injection at the concentration of 120 mg/mL. Mechanistically, JMB2005 could bridge tumor cells and T cells with both Fab arms and promote T-cells to function as direct tumor cell killers. It could also promote T cell activation by blocking the binding of PD-L1 to CD80. Furthermore, JMB2005 has exhibited a favorable half-life and has demonstrated promising anti-tumor therapeutic efficacy in vivo. Conclusion Consequently, the present study showed that the novel humanized common light chain bispecific antibody JMB2005 may represent a novel therapeutic agent of great clinical potential.

Engineering and physicochemical characterization of a novel, stable, symmetric bispecific antibody with dual target-binding using a common light chain.

Bispecific antibodies (BsAbs) have emerged as a major class of antibody therapeutics owing to their substantial potential in disease treatment. While several BsAbs have been successfully approved in recent years, ongoing development efforts continue to focus on optimizing various BsAbs tailored to particular antigens and action mechanisms, aiming to achieve favorable physicochemical properties. BsAbs generally encounter challenges due to their unfavorable physicochemical characteristics and poor manufacturing efficiencies, highlighting the need for optimization to achieve reliable productivity and developability. Herein, we describe the development of a novel symmetric BsAb, REGULGENT™ (N-term/C-term), comprising two Fab domains, using a common light chain. The heavy chain fragment encoded two antigen-binding determinants in one chain. The design and production of REGULGENT™ (N-term/C-term) are simple owing to the use of the same light chain, which does not induce heavy and light chain mispairing, frequently observed with the asymmetric BsAb format. REGULGENT™ (N-term/C-term) exhibited high expression and low aggregation characteristics during cell culture and stress treatment under low pH conditions. Differential scanning calorimetric data indicated that REGULGENT™ molecules had high conformational stability, similar to that of stabilized monoclonal antibodies. Surface plasmon resonance data showed that REGULGENT™ (N-term/C-term) could bind to two antigens simultaneously and exhibited a high affinity for two antigens. In summary, the symmetric BsAb format of REGULGENT™ confers its desirable IgG-like physicochemical properties, thus making it an excellent candidate for commercial development. The findings demonstrate a novel BsAb with substantial development potential for clinical applications.

Petosemtamab (MCLA-158) with pembrolizumab as first-line (1L) treatment of recurrent/metastatic (r/m) head and neck squamous cell carcinoma (HNSCC): Phase 2 study.

6014 Background: EGFR is a known oncogenic driver in HNSCC, and the leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is a receptor expressed on cancer stem cells, including in HNSCC. Petosemtamab is a human, common light chain, IgG1 bispecific antibody with ADCC-enhanced activity, targeting EGFR and LGR5. In the dose escalation part of a phase 1/2 study, the recommended phase 2 dose (RP2D) was determined to be 1500 mg every 2 weeks (Q2W). Interim data of petosemtamab monotherapy at the RP2D in 2L/3L HNSCC led to a 37.2% overall response rate (ORR; per investigator) with 6.0 months (mo) median duration of response (DOR) [Cohen, Cancer Research 2023]. Petosemtamab (RP2D) with pembrolizumab (400 mg Q6W) is being investigated in an expansion cohort of the ongoing phase 2 study (NCT03526835) in 1L HNSCC. Methods: Primary endpoints are safety and investigator-assessed ORR (RECIST v1.1). Secondary endpoints include DOR, progression-free survival (per investigator), and overall survival. Key eligibility criteria were r/m HNSCC with no prior systemic therapy, PD-L1 combined positive score ≥1, ECOG PS 0–1, measurable disease, and primary tumor location in oropharynx (regardless of p16 status), oral cavity, hypopharynx, or larynx. Results: No dose-limiting toxicities were observed in the safety run-in. As of a November 6, 2023 data cutoff, 26 pts were treated (24 continuing therapy at the data cutoff) and median follow-up was 1.35 mo. Median age was 62.5 years (range 23–80), ECOG PS 0/1 in 10/16 pts, and 65.4% were male. The most frequent primary tumor locations were oropharynx (34.6%), oral cavity (19.2%), and hypopharynx (19.2%). A median of 2 cycles (range 1–8) were administered. The combination was well tolerated and no significant overlapping toxicities were observed. Treatment-emergent adverse events (AEs) were reported in 26 pts, and most were Grade (G) 1 or 2 in severity (no G4–5 were observed). The most frequent AEs (all Gs/G3) were acneiform dermatitis (30.8%/0%), asthenia (26.9%/0%), and rash (26.9%/0%). Infusion-related reactions (composite term) were reported in 26.9% (all Gs) and 3.8% (G3) of pts, all occurred during the first infusion and resolved. Among 10 pts evaluable for efficacy (≥2 cycles and ≥1 post-baseline scan, or early progressive disease), there were 1 confirmed complete response, 2 confirmed and 3 unconfirmed partial responses (2 confirmed after data cutoff), 3 stable disease, and 1 progressive disease; with all 6 responders on treatment at data cutoff. Enrollment has been completed and updated data will be presented. Conclusions: Petosemtamab, a first-in-class EGFR x LGR5 bispecific antibody, in combination with pembrolizumab demonstrates a well-tolerated safety profile and promising preliminary clinical efficacy as 1L treatment for pts with r/m HNSCC. Clinical trial information: NCT03526835 .

Efficacy and safety of MCLA-129, an EGFR/c-MET bispecific antibody, in advanced non-small cell lung cancer (NSCLC).

8604 Background: MCLA-129 is a common light chain, Biclonics, bispecific antibody targeting EGFR and c-MET with enhanced antibody-dependent cellular cytotoxicity. Here we describe the ongoing first-in-human phase 1/2 study of MCLA-129 in patients with NSCLC, including patients with MET exon 14 skipping (METex14) mutation (cohort A), EGFR exon 20 insertion (exon20ins)-mutated (cohort B) and sensitized EGFR-mutated disease (cohort C). Methods: Patients received MCLA-129 (100–2000 mg) IV biweekly or 1000-1500 mg IV weekly in a 28-day cycle in dose escalation. Doses at 1500 and 2000 mg IV biweekly are being explored in dose expansion. The primary endpoint was safety and tolerability. Objective response rate by investigator assessment per RECIST v1.1 of patients with NSCLC treated at 1500 and 2000 mg IV biweekly in three cohorts is reported. Results: As of 26 May 2023, 217 enrolled patients received MCLA-129 in China. The data cut-off was 24 Nov 2023. The most common treatment-emergent adverse events (TEAEs) were infusion related reaction (71.9%), hypoalbuminemia (54.8%), decreased neutrophil count (46.1%), and decreased white blood cell count (40.1%). TEAEs mostly were grade 1-2. Grade ≥3 TEAEs and drug-related TEAEs were reported in 51.6% and 39.2% of patients, respectively. 176 of the 215 NSCLC patients were evaluable. The confirmed objective response rates (ORR) in Cohort A, B and C were 43.5%, 28.6% and 21.8%, respectively. Especially, for patients in Cohort A receive prior MET TKI, the ORR was 37.5% and disease control rate (DCR) was 93.8%. Overall efficacy is shown (Table). Conclusions: MCLA-129 demonstrates robust and durable antitumor activity in patients with NSCLC harboring METex14, EGFR exon20ins and sensitized EGFR mutations, with a manageable safety profile; enrollment in dose expansion is ongoing. Clinical trial information: NCT04930432 . [Table: see text]

Phase I study of MCLA-145, a bispecific antibody targeting CD137 and PD-L1, in solid tumors, as monotherapy or in combination with pembrolizumab.

2520 Background: MCLA-145, a human common light chain bispecific antibody targeting CD137 and the PD-1/PD-L1 axis, is designed to enhance both antigen-mediated T cell activation via CD137 costimulation, and blockade of inhibitory PD-L1. Interim data from the ongoing phase 1 study (NCT03922204) are presented. Methods: Patients (pts) with PD-L1 ≥1% advanced/metastatic solid tumors received MCLA-145 IV as monotherapy Q2W/Q3W in 21/28-d cycles respectively, or in combination with pembrolizumab 200 mg Q3W in 21-d cycles. Pts enrolled in combination had cancers that either relapsed after PD-(L)1 therapies or were immunotherapy naïve. Primary objectives are safety, tolerability and dose-limiting toxicity (DLT) of MCLA-145 alone or combined with pembrolizumab, and determination of the recommended dose for expansion (RDE). Secondary endpoints include efficacy, pharmacokinetics, pharmacodynamics (PD) and immunogenicity. Results: As of a December 4, 2023 data cutoff, 72 pts with 26 cancer types were treated; 25% of pts had non-small cell lung cancer (NSCLC). 3 pts were continuing combination therapy. Monotherapy: 53 pts (median age 60 y, 49% male) were treated across 8 dose levels (47 pts 0.4-75 mg Q2W, 6 pts 40 mg Q3W). Median number of cycles was 2 (range 1-39). 6 pts had DLTs in the 25-75 mg dose range (febrile neutropenia [2 pts], hemolytic anemia, myositis, ALT/AST increase, neutrophil/platelet decrease [1 pt each]). Most common adverse events (AEs; all grades/G3-4) were fatigue (51%/4%), decreased appetite (34%/2%), dyspnea (32%/0%), anemia (30%/9%), ALT/AST increase (25%/11%), and pyrexia (25%/0%). The incidence of any G3-4 AE was lower with 40 mg Q3W than across all Q2W dose levels (33% vs 66%). Combination: 19 pts (median age 61 y, 47% male) were treated with MCLA-145 10, 25 or 40 mg plus Q3W. Median number of cycles was 5 (range 1-16). No DLTs occurred. Most common AEs (all grades/G3-4) were fatigue (58%/11%), cough (42%/0%), constipation (32%/0%) and ALT/AST increase (21%/11%). The RDE was established at 40 mg Q3W for both monotherapy and combination. Preliminary antitumor activity was observed with monotherapy (52 evaluable pts): 5 partial responses (PRs) in glioblastoma (lasting >3 y), sarcoma, cervical, anal, and gastric cancer (treated for 2-11 mo); and combination (19 evaluable pts): 1 PR in Merkel cell carcinoma (treated 12+ mo), 1 complete response in PD-L1+ NSCLC (treated 6+ mo), both after prior immunotherapy. Disease control rate was 37% with monotherapy and 68% with combination. Exposure was dose-dependent with a terminal half-life of 69 h at 75 mg. Measure of peripheral blood Ki67+ CD8 T cells supports maximal PD activity at 40 mg. Conclusions: MCLA-145 given alone or in combination with pembrolizumab had a well-tolerated and manageable safety profile with encouraging clinical activity, including in pts who relapsed after PD-(L)1 therapies. Clinical trial information: NCT03922204 .

A novel B7-H3 x CD3 bispecific T-cell engager for the treatment of B7-H3+ tumors.

e14509 Background: B7-H3 (CD276) is a transmembrane protein highly expressed in solid tumors but with limited expression in normal tissues. Its presence is strongly associated with the inhibition of T-cell activity, cancer progression, and invasion. Furthermore, B7-H3 is strongly associated with poor prognosis and reduced patient survival. Given its crucial role in tumor biology, B7-H3 has emerged as an attractive therapeutic target for drug development. We utilized ExMab heterodimer platform and common light chain technology to develop EX105, a next-generation B7-H3×CD3 bispecific antibody. This innovative T-cell engager exhibits excellent anti-tumor activity in mouse models and good safety profile in NHP tox study. Methods: EX105 was generated using ExMab heterodimer platform and common light chain technology to form a heterodimeric IgG1 structure. Preclinical pharmacology studies, efficacy, and safety were evaluated. Anti-tumor activity study of EX105 includes in vitro T-cell cytotoxicity assays and in vivo PBMC reconstituted xenograft mice models. Preclinical safety evaluation of EX105 was performed through GLP single-dose and repeat-dose tox studies in non-human primates (NHPs). Results: Based on ELISA and FACS analyses, EX105 can bind to both human and cynomolgus monkey B7-H3 and CD3 antigens. Furthermore, SPR data suggest that EX105 exhibits a high affinity to B7-H3 and a much lower affinity to CD3. In consistent with that, EX105 only induced relatively low cytokine release. Nevertheless, EX105 showed a potent in vitro T cell cytotoxicity against B7-H3+ tumor cell lines. The in vivo anti-tumor activity of EX105 was further determined in several tumor xenograft models. Treatment with EX105 resulted in remarkable tumor regression in both “hot” tumor model and “cold” tumor model, indicating its effectiveness across a range of tumor types. Tox study in NHPs showed minimal adverse effects and no liver impairment after EX105 administration, even at high doses. The cytokine release data observed in this study suggests a good safety profile of EX105, with much lower cytokine release compared to other T cell engagers. Conclusions: These findings collectively indicate that EX105 has the potential to become a promising therapeutic agent for the treatment of various B7-H3 positive cancers. The first-in-human clinical trial for patients with advanced B7-H3 positive cancers is due to start.

Abstract LB127: A first-in-class bispecific ADC targeting FORL1 and MUC1 exhibits promising anti-tumor activity in a FOLR1low xenograft model

Abstract Folate receptor alpha (FOLR1) is specifically overexpressed in malignant tumors of epithelial origin, including ovarian, lung and breast cancers. While antibody-drug conjugate (ADC) therapies targeting FOLR1 have shown promise in clinical trials, there is still a large proportion of patients, particularly those with low FOLR1 expression, who do not respond, indicating new therapeutic strategies are needed. MUC1 is another tumor-associated antigen that is likewise overexpressed in epithelial ovarian cancer, breast cancer, and NSCLC, among other cancers; analysis of ovarian PDX tumors by immunohistochemistry (IHC) suggests that FOLR1 and MUC1 are both highly expressed in this cancer type. We therefore hypothesized that dual targeting of FOLR1 and MUC1 could provide therapeutic benefit in ovarian cancer as well as a broad range of tumors. We generated a FOLR1 x MUC1 bispecific antibody (bsAb) from anti-FOLR1 and anti-MUC1 monoclonal antibodies that were produced using RenLite® fully human common light chain mice. These parental monoclonal antibodies recognize both human and cynomolgus monkey antigens. Anti-FOLR1 antibodies were highly specific for FOLR1, as they did not recognize other FOLR family members. The anti-MUC1 antibody was previously confirmed to target MUC1-C, the juxtamembrane domain of MUC1, ensuring tumor cell recognition regardless of MUC1 cleavage. Assessment of the novel FORL1 x MUC1 bsAb indicates binding to several ovarian, breast and NSCLC cell lines, and shows enhanced internalization in MDA-MB-468 and T47D cell lines when compared to parental antibodies. Subsequently, the FORL1 x MUC1 bsAb was conjugated to vcMMAE with a DAR of 4, to generate a bispecific ADC, BCG023. BCG023 exhibited superior tumor growth inhibition compared to benchmark ADCs conjugated with the same payload and DAR in a low FOLR1-expressing NSCLC PDX model. These data suggest that targeting FOLR1 and MUC1 with a bispecific ADC is a promising therapeutic strategy for the treatment of ovarian and other tumors. Citation Format: Yifu Zhang, Chengzhang Shang, Chaoshe Guo, Yi Yang. A first-in-class bispecific ADC targeting FORL1 and MUC1 exhibits promising anti-tumor activity in a FOLR1low xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB127.

Abstract 2379: The bispecific antibody KA-3007 against B7H3 and CD28 enhances T cell activation in solid tumors

Abstract Immunotherapy, revolutionizing the treatment of advanced solid tumors by blocking PD1 signaling, faces challenges as most cancers remain unresponsive to anti-PD-1 therapy. CD28, a key immune co-stimulatory receptor on T cells, often lacks ligands expressed on tumor cells. B7H3 is highly expressed in various solid tumors. In this study, we developed a bispecific antibody (BsAb) named KA-3007, targeting B7H3 and human CD28 to activate T cells in the tumor microenvironment. KA-3007, featuring an identical light chain, was developed using our common light chain bispecific antibody discovery platform with knob-into-hole technology. This BsAb is in regular IgG1 format with mutations in the Fc domain to eliminate ADCC, ADCP, and CDC activity. Its two Fabs bind to B7H3 with high affinity and CD28 with relatively low affinity. Co-culturing T cells with tumor cells expressing B7H3 enhances T cell activation. In PBMC humanized immunodeficient mouse tumor models, KA-3007 effectively suppresses the growth of NCI-H292 tumors, with or without a PD1 antagonist antibody. Furthermore, this antibody exhibits a half-life of more than five days in the blood of Balb/c mice. Finally, we assessed the developability of KA-3007 with different treatments such as high concentration, low pH, repeated freezing and thawing, and high temperature, and it shows neither aggregation nor degradation. In conclusion, the BsAb KA-3007, developed with our common light chain antibody discovery platform, emerges as a promising pre-clinical candidate drug for the treatment of solid cancers. Citation Format: Guojin Wu, Zhengcheng Guo, Hao Peng, Feng Hao, Feng He, Jinying Ning. The bispecific antibody KA-3007 against B7H3 and CD28 enhances T cell activation in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2379.

Abstract 6366: The triple-specific antibody KA-3009 against EGFR, B7H3, and 4-1BB specifically enhances T cell activity within the tumor

Abstract Cancer immunotherapy with 4-1BB agonists faces limited further clinical development due to dose-limiting toxicity. Moreover, EGFR and B7H3 are highly expressed in various solid tumors, as well as in different normal tissues. In this study, we developed a trispecific antibody, KA-3009, targeting EGFR, B7-H3, and human 4-1BB, with the aim of restricting 4-1BB stimulation within tumors. KA-3009 was developed based on our common light chain bispecific antibody discovery platform and nanobody antibody discovery platform using knob-into-hole technology. It is in IgG1 format with mutations in the Fc domain to eliminate ADCC, ADCP, and CDC activity. Featuring two identical nanobodies at the C-terminal against 4-1BB, KA-3009 binds to EGFR and B7H3 respectively with two Fabs at the N-terminal, sharing an identical light chain. KA-3009 exhibits low avidity binding to tumor cells expressing only EGFR or B7H3, while binding to tumor cells co-overexpressing EGFR and B7H3 with increased avidity by more than ten times. Additionally, KA-3009 binds to 4-1BB expressed on 293T cells and activated human T cells. In the presence of a low concentration of CD3 antibody to stimulate T cell activation, co-culture of tumor cells co-expressing EGFR and B7H3 with primary T cells isolated from human PBMC enhances T cell activation and proliferation. In PBMC humanized immunodeficient mouse tumor models, KA-3009 effectively suppresses the growth of NCI-H1568 tumors expressing both EGFR and B7H3 but does not affect the growth of MS751 and NCI-H661 tumors, which only express EGFR or B7H3, respectively. Moreover, KA-3009 has a half-life of more than five days in the blood of Balb/c mice. Finally, we assessed the developability of this antibody with different treatments such as high concentration, low pH, repeated freezing and thawing, and high temperature, and KA-3009 shows neither aggregation nor degradation. In conclusion, the triple-specific antibody KA-3009, developed with our common light chain antibody discovery platform and nanobody antibody discovery platform, is a promising pre-clinical candidate drug for the treatment of different solid cancers that co-overexpress EGFR and B7H3. Citation Format: Guojin Wu, Tongtong Liu, Jiabei Liang, Hao Peng, Feng Hao, Jinying Ning. The triple-specific antibody KA-3009 against EGFR, B7H3, and 4-1BB specifically enhances T cell activity within the tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6366.

Abstract 2378: The triple-specific antibody KA-3003, targeting PSMA, CD28, and 4-1BB, is developed for the treatment of prostate cancer by enhancing T cell activity within the tumor

Abstract Immunotherapy, revolutionizing the treatment of advanced solid tumors by blocking PD1 signaling, has encountered a challenge in prostate cancer, exhibiting a low response ratio to anti-PD-1 therapy. Furthermore, cancer immunotherapy using 4-1BB agonists faces limited clinical development due to dose-limiting toxicity. Here, we developed a tri-specific antibody, KA-3003, targeting PSMA, CD28, and 4-1BB, with the goal of enhancing T cell activation in the tumor microenvironment. This antibody was created using our common light chain bispecific antibody discovery platform and nanobody antibody discovery platform with knob-into-hole technology. KA-3003, in IgG1 format, features mutations in the Fc domain to eliminate ADCC, ADCP, and CDC activity. It contains two identical nanobodies at the C-terminal against 4-1BB, while binding to PSMA and CD28 with two Fabs at the N-terminal, sharing an identical light chain. KA-3003 exhibits high affinity for PSMA and relatively lower affinity for CD28 and CD137. In the presence of a low concentration of CD3 antibody to stimulate T cell activation, co-culture of tumor cells expressing PSMA with primary T cells isolated from human PBMC enhances T cell activation and proliferation. In PBMC humanized immunodeficient mouse tumor models, KA-3003 effectively suppresses the growth of 22Rv1 tumors with or without a PD1 antagonist antibody. Additionally, KA-3003 has a half-life of more than five days in the blood of Balb/c mice. Finally, we assessed the developability of this antibody with different treatments such as high concentration, low pH, repeated freezing and thawing, and high temperature. KA-3003 shows neither aggregation nor degradation. In conclusion, the tri-specific antibody KA-3003, developed with our common light chain antibody discovery platform, stands out as a promising pre-clinical candidate drug for the treatment of prostate cancer. Citation Format: Guojin Wu, Zhengcheng Guo, Hao Peng, Feng Hao, Tongtong Liu, Jinying Ning. The triple-specific antibody KA-3003, targeting PSMA, CD28, and 4-1BB, is developed for the treatment of prostate cancer by enhancing T cell activity within the tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2378.

Abstract 2382: KY-CLC-mouse: a common light chain bispecific antibody discovery platform

Abstract Bispecific antibodies (BsAbs) have emerged as a potent therapeutic tool in cancer treatment, gaining increasing centrality in therapeutic applications. However, the development of bispecific antibodies is hindered by the mispairing of light and heavy chains. In response to this challenge, our company developed the KY-CLC-mouse, featuring an identical κ light chain without the expression of the λ light chain. This mouse shows great promise in bispecific antibody development, as the identical κ light chain, modified from a frequently occurring light chain in mice, pairs with multiple heavy chains. The advantages of such a light chain include good solubility and stability, as well as ease of humanization without post-translational modifications in the complementary-determining regions (CDRs). Furthermore, the CDRs were modified to reduce immunogenicity. This KY-CLC-mouse platform has facilitated the identification of antibodies that specifically target over a dozen antigens, such as PD-1, LAG33, MET, PSMA, and CD28, with KD ranging from 0.1 nM to 10 nM. We apply the knob-in-hole technology to assemble bispecific antibodies, targeting two antigens or different epitopes of the same antigen, following their humanization. Prime examples include fully humanized BsAbs PD1xLAG-3, PDL1xEGFR, PSMAxCD28, and EGFRxB7H3. These bispecific antibodies have an identical light chain and were developed with knob-into-hole technology. With different treatments such as high concentration, low pH, repeated freezing and thawing, and high temperature, the BsAbs show neither aggregation nor degradation, suggesting good developability of these antibodies. Additionally, these BsAbs have a half-life of more than five days in the blood of both mice and monkeys. In conclusion, our common light chain KY-CLC-mouse is a powerful bispecific antibody discovery platform. Citation Format: Hao Peng, Guojin Wu, Feng Hao, Feng He, Yi Gu, Jinying Ning. KY-CLC-mouse: a common light chain bispecific antibody discovery platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2382.

Abstract 2381: The bispecific antibody KA-3001, targeting PSMA and CD28, specifically enhances T cell activation within prostate tumors

Abstract Immunotherapy, which involves blocking PD-1 signaling, has revolutionized the treatment paradigm for advanced solid tumors. However, prostate cancer exhibits a low response ratio to anti-PD-1 therapy. In this study, we have developed a bispecific antibody (BsAb), KA-3001, targeting PSMA and human CD28, aiming to enhance T cell activation in the tumor microenvironment. KA-3001, in a regular IgG1 format, features an identical light chain and was designed using our common light chain bispecific antibody discovery platform with knob-into-hole technology. Mutations in the Fc domain were introduced to eliminate ADCC, ADCP, and CDC activity. KA-3001's two Fabs demonstrate high affinity for PSMA and relatively low affinity for CD28. In comparison to REGN5678, KA-3001 exhibits significantly stronger binding to PSMA-expressing prostate tumor cells, along with relatively lower CD28 activation, minimizing toxicity while maintaining anti-tumor efficacy. In PBMC humanized immunodeficient mouse tumor models, KA-3001 effectively suppresses the growth of 22Rv1 tumors with or without PD-1 antagonist antibody. Furthermore, KA-3001 has a half-life of more than five days in the blood of Balb/c mice. Finally, we assessed the developability of this antibody through various treatments such as high concentration, low pH, repeated freezing and thawing, and high temperature. The BsAb demonstrates neither aggregation nor degradation. In conclusion, the BsAb KA-3001, developed using our common light chain antibody discovery platform, stands out as a promising pre-clinical candidate drug for the treatment of prostate cancer. Citation Format: Jiabei Liang, Chen Su, Hao Peng, Feng Hao, Tongtong Liu, Jinying Ning, Guojin Wu. The bispecific antibody KA-3001, targeting PSMA and CD28, specifically enhances T cell activation within prostate tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2381.

Abstract 2617: BCG022, a novel HER3 × MET bispecific antibody-drug conjugate (bsADC), demonstrates promising anti-tumor efficacy

Abstract HER3 and MET expression act as bypass resistance mechanisms, conferring resistance to multiple therapeutic agents, such as EGFR TKI treatment. HER3 and MET are also co-expressed at high prevalence in multiple tumor types, including gastric, colorectal, breast, and non-small-cell lung cancer (NSCLC). In addition, HER3 and MET are frequently overexpressed in liver metastases from patients with colorectal cancer, indicating that targeting both targets may provide clinical benefit. We previously reported generation of a fully human bispecific antibody (bsAb) targeting HER3 and MET using RenLite® mice, which contain the full human heavy chain variable domain with a common human kappa light chain to facilitate bispecific antibody assembly. This bsAb demonstrated reactivity to human and cynomolgus monkey antigens and binding to multiple cancer cell lines, including NSCLC, gastric, colorectal, and hepatocellular cancer cell lines. The bsAb also showed enhanced internalization activity compared to parental monovalent antibodies in the NUGC-4 cell line expressing HER3 and MET. Here, we evaluated the efficacy of the bsAb when conjugated to two distinct payloads: vcMMAE and BLD1102, Biocytogen’s novel, proprietary linker/payload system composed of a DNA Topo I inhibitor payload (BCPT02) and a highly hydrophilic protease-cleavable linker. The in vivo efficacy of BCG022-vcMMAE and BCG022-BLD1102 was evaluated in cell-derived NSCLC and gastric cancer xenografts, and in patient-derived gastric and pancreatic xenograft models. Both demonstrated strong anti-tumor activity, with efficacy greater than or comparable to benchmark ADCs. Taken together, these promising preclinical results suggest that the BCG022 bsADC could be a novel treatment for HER3 and MET co-expressing tumors. Citation Format: Zhuolin Li, Chengzhang Shang, Zhenyan Han, Gao An, Chaoshe Guo, W. Frank An, Yi Yang. BCG022, a novel HER3 × MET bispecific antibody-drug conjugate (bsADC), demonstrates promising anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2617.

Abstract 2619: A novel EGFR × HER3-targeting bispecific antibody drug-conjugate, BCG019, demonstrates robust anti-tumor efficacy in preclinical evaluation

Abstract EGFR and HER3 are receptor tyrosine kinases that are highly expressed in multiple epithelial tumors. EGFR point mutations and small insertions within the kinase domain are common in lung cancer. HER3 is overexpressed in patients who are resistant to EGFR-TKI therapy, and is one of the common resistance mechanisms of EGFR and HER2-targeted therapy. We hypothesize that simultaneous targeting of EGFR and HER3 with a bispecific ADC will have the potential to overcome the resistance caused by HER3 after EGFR-targeted therapy and obtain better efficacy than the combination of EGFR monotherapy and HER3 monotherapy. We first generated fully human bispecific antibodies (bsAbs) targeting EGFR and HER3 using RenLite® mice, which contain the full human heavy chain variable domain with a common human kappa light chain to facilitate bispecific antibody assembly. The anti-EGFR × HER3 bsAb demonstrated reactivity to human and cynomolgus monkey antigens and binding to multiple cancer cell lines, including NSCLC, gastric, pancreatic, colorectal, and breast cancer cell lines. The anti-EGFR × HER3 bsAb also showed high internalization activity in cell lines expressing EGFR and HER3. The anti-EGFR × HER3 bsAb was then conjugated with vc-MMAE (valine-citrulline-monomethyl auristatin E) for proof-of-concept studies, as well as BLD1102, Biocytogen’s novel, proprietary linker/payload system composed of a DNA Topo I inhibitor payload (BCPT02) and highly hydrophilic protease-cleavable linker, to generate EGFR- and HER3-targeting bispecific ADC candidates BCG019. In vivo efficacy of the bsADC candidates were evaluated in cell-derived NSCLC and gastric cancer xenografts and in patient-derived gastric and colorectal xenograft models. Both bsADCs showed superior anti-tumor activity when compared to benchmark ADCs. Collectively, these results suggest that our novel anti-EGFR × HER3 bsADC has therapeutic potential for EGFR and HER3 co-expressing tumors. Citation Format: Zhuolin Li, Chengzhang Shang, Gao An, Chaoshe Guo, W. Frank An, Yi Yang. A novel EGFR × HER3-targeting bispecific antibody drug-conjugate, BCG019, demonstrates robust anti-tumor efficacy in preclinical evaluation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2619.

Abstract 2621: Preclinical Efficacy of DM002, a bispecific HER3 × MUC1 antibody-drug conjugate with a novel DNA topoisomerase I inhibitor, in solid tumor models

Abstract MUC1 is a known tumor-associated antigen (TAA), but previous anti-MUC1 agents have shown limited clinical efficacy due to neutralization by the soluble N-terminal autoproteolytic product of MUC1. More recently, HER3-targeted ADCs, such as Patritumab deruxtecan, have shown encouraging early clinical activity in a variety of tumors. However, the majority of patients did not exhibit response to paritumab deruxtecan treatment. Therefore, improved therapies targeting these TAAs are needed. As HER3 and MUC1 are co-expressed in a wide range of cancer types, we hypothesized that simultaneous targeting of HER3 and MUC1 with a bispecific antibody-drug conjugate (bsADC) could potentially lead to increased efficacy, reduced resistance and improved safety in the clinical setting. We generated an anti-HER3 × MUC1 bispecific antibody using the fully human common light chain antibody transgenic mice (RenLite®). The HER3 × MUC1 bsAb showed improved cell binding and internalization in vitro compared to the parental mAb, suggesting synergy between the two arms. We then conjugated the HER3xMUC1 bsAb with vcMMAE for proof-of-concept studies, or with our novel DNA topoisomerase I inhibitor linker/payload conjugates (BLD1102) to make DM002-BLD1102 bsADC. Both bsADCs potently inhibited the growth of HER3+MUC1+ PDX tumors. DM002-vcMMAE also showed greater in vivo efficacy than its parental mAb ADCs, consistent with their in vitro internalization activity. DM002-BLD1102 also showed strong anti-tumor activity in DM002-vcMMAE resistant PDX models, suggesting the superiority of this new DNA topoisomerase I inhibitor linker/payload over the classical vcMMAE linker/payload. In conclusion, the HER3 × MUC1 bsAb is a promising agent for the treatment of HER3 and MUC1 positive tumors. Citation Format: Yifu Zhang, Chengzhang Shang, Nannan Wang, Gao An, Chaoshe Guo, W. Frank An, Yi Yang. Preclinical Efficacy of DM002, a bispecific HER3 × MUC1 antibody-drug conjugate with a novel DNA topoisomerase I inhibitor, in solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2621.

Abstract 6363: The biparatopic antibody KA-3005 against MET is suitable for developing antibody-drug conjugates (ADCs) to treat MET-expressing cancers

Abstract MET is overexpressed in a significant fraction of lung (25%-50%) and gastroesophageal cancers, making the targeting of MET with an antibody-drug conjugate (ADC) an attractive approach. Here, we developed a bispecific antibody (BsAb) named KA-3005, targeting two different epitopes within the extracellular domain of MET using our common light chain antibody discovery platform. KA-3005 has a regular antibody structure with an identical light chain, developed using knob-into-hole technology. This antibody exhibits significantly higher avidity to MET expressed on tumor cells, with an EC50 of less than 0.1nM compared to its parent antibodies. It also completely blocks the binding of HGF to MET and the MET downstream pathway at a concentration as low as 1nM. This biparatopic antibody demonstrates a faster internalization rate upon binding to the surface of tumor cells expressing MET than its parent antibodies. KA-3005 effectively suppressed the growth of HCC827-HGF tumors in a xenograft mouse model. Additionally, it has a half-life of more than five days in the blood of Balb/c mice. Finally, we assessed the developability of KA-3005 under different treatments such as high concentration, low pH, repeated freezing and thawing, and high temperature, and it showed neither aggregation nor degradation. In conclusion, this biparatopic antibody, KA-3005, developed with our common light chain antibody discovery platform, is stable and holds potential for the development of ADCs targeting MET. Citation Format: Guojin Wu, Jiabei Liang, Hao Peng, Feng Hao, Tongtong Liu, Jinying Ning. The biparatopic antibody KA-3005 against MET is suitable for developing antibody-drug conjugates (ADCs) to treat MET-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6363.

Abstract 6364: The bispecific antibody KA-3006 against both EGFR and MET surpasses amivantamab in efficacy

Abstract A bispecific antibody (BsAb) like amivantamab, which targets both the epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition factor (MET) by blocking downstream pathways, has been proven to overcome resistance to targeted therapies, including EGFR inhibitors, in patients with various cancers. In this study, we developed a bispecific antibody, KA-3006, targeting both EGFR and MET using our common light chain antibody discovery platform. KA-3006 has a regular antibody structure with an identical light chain, developed through knob-into-hole technology. It exhibits high affinity for MET and relatively low affinity for EGFR. KA-3006 specifically blocks both the binding of EGF to EGFR and HGF to MET with higher efficiency than amivantamab, resulting in the blockade of downstream signaling from EGFR and MET. In comparison to amivantamab, KA-3006 demonstrates a faster internalization rate when binding to the surface of tumor cells expressing both EGFR and MET. KA-3006 showed superior antitumor activity over amivantamab in the HCC827-HGF in vivo model. Additionally, KA-3006 has a half-life of more than five days in the blood of Balb/c mice. Finally, we assessed the developability of this BsAb with different treatments such as high concentration, low pH, repeated freezing and thawing, and high temperature, and KA-3006 showed neither aggregation nor degradation. In conclusion, the BsAb KA-3006, developed with our common light chain antibody discovery platform, is a promising pre-clinical candidate drug for the treatment of various solid cancers with overactivated EGFR and MET signaling, and it holds potential for developing ADCs targeting both EGFR and MET. Citation Format: Guojin Wu, Jiabei Liang, Hao Peng, Feng Hao, Tongtong Liu, Jinying Ning. The bispecific antibody KA-3006 against both EGFR and MET surpasses amivantamab in efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6364.

Abstract 6365: The bispecific antibody KA-3008, targeting both EGFR and B7H3, increases internalization and minimizes on-target, off-tumor toxicity

Abstract EGFR and B7H3 are highly expressed in various solid tumors, as well as in different normal tissues. Targeting EGFR or B7H3 alone with inhibitory monoclonal antibodies or antibody-drug conjugates (ADCs) may result in on-target, off-tumor toxicity. In this study, we developed a bispecific antibody (BsAb) named KA-3008, targeting both EGFR and B7H3 using our common light chain antibody discovery platform. KA-3008, in IgG1 format, features a regular antibody structure with an identical light chain, developed through knob-into-hole technology. Compared to its parent antibodies, its affinities for both EGFR and B7H3 decrease by more than ten times. It binds to cell lines expressing EGFR or B7H3 alone with an EC50 of more than 10nM, while it binds to cell lines expressing both EGFR and B7H3 with an EC50 of less than 1nM. KA-3008 also blocks the binding of EGF to EGFR and increases internalization when binding to both EGFR and B7H3 compared to binding to EGFR or B7H3 alone. In human tumor mouse models, KA-3008 effectively suppresses the growth of NCI-H1568 tumors expressing both EGFR and B7H3, but does not affect the growth of MS751 tumors that only highly express EGFR with low expression of B7H3. Additionally, KA-3008 has a half-life of more than five days in the blood of Balb/c mice. Finally, we assessed the developability of this BsAb with different treatments such as high concentration, low pH, repeated freezing and thawing, and high temperature, and KA-3008 shows neither aggregation nor degradation. In conclusion, the BsAb KA-3008, developed with our common light chain antibody discovery platform, is stable and holds potential for the development of ADCs targeting both EGFR and B7H3. Citation Format: Guojin Wu, Li Ding, Hao Peng, Feng Hao, Feng He, Jinying Ning. The bispecific antibody KA-3008, targeting both EGFR and B7H3, increases internalization and minimizes on-target, off-tumor toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6365.

Abstract 2616: BCG033, a novel bispecific antibody-drug conjugate targeting PTK7 and TROP2, demonstrates preclinical efficacy against triple-negative breast cancer and other solid tumor xenografts

Abstract Metastatic triple-negative breast cancer (TNBC) patients have poor overall survival, highlighting the need for novel treatments. Although the TROP2-targeting ADC sacituzumab govitecan recently received accelerated approval from the FDA for the treatment of patients with metastatic TNBC, the on-target toxicity of this single-target agent has limited clinical efficacy. We sought to improve the specificity and efficacy of future TNBC-targeted therapies by generating a bispecific antibody-drug conjugate (bsADC) targeting TROP2 and PTK7, another tumor-associated antigen (TAA) that is highly expressed in TNBC and correlates with poor prognosis and metastatic disease. We previously generated fully human antibodies targeting PTK7 and TROP2 from Biocytogen’s fully human, common light chain antibody transgenic RenLite® mice, which were selected such that their monovalent antibodies exhibited reduced internalization profiles for improved tumor selectivity. These antibodies were then constructed into a bispecific antibody targeting PTK7 × TROP2, which demonstrated reactivity to human and cynomolgus monkey antigens and binding to multiple cancer cell lines, including TNBC, with high affinity. PTK7 × TROP2 bsAb also showed enhanced internalization in vitro compared with parental monovalent antibodies. Next, the PTK7 x TROP2 bsAb was conjugated to vcMMAE with a drug-to-antibody ratio (DAR) of 4 to generate a bsADC (BCG033). BCG033-vcMMAE demonstrated superior activity to benchmark and parental ADCs in a cell line-derived xenograft (CDX) TNBC model with low PTK7 expression. Here, we demonstrate that BCG033-vcMMAE ADCs also exhibited superior efficacy to benchmark ADCs in PDX models, including TNBC xenografts and NSCLC xenografts. We next tested the efficacy of the PTK7 x TROP2 bsAb conjugated to BLD1102, Biocytogen’s novel, proprietary linker/payload composed of a DNA Topo I inhibitor payload and a highly hydrophilic protease-cleavable linker, with a DAR of 8. BCG033-BLD1102 showed potent activity in a colorectal cancer PDX model in which benchmark ADCs were inactive, even at high doses. In summary, BCG033 conjugates demonstrate promising preclinical efficacy in vivo, which ultimately could provide a new treatment option for TNBC and other solid tumors expressing PTK7 and TROP2. Citation Format: Sufei Yao, Chengzhang Shang, Gao An, Chaoshe Guo, W. Frk an, Yi Yang. BCG033, a novel bispecific antibody-drug conjugate targeting PTK7 and TROP2, demonstrates preclinical efficacy against triple-negative breast cancer and other solid tumor xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2616.

Abstract 2724: BCG020, a novel bispecific antibody targeting EGFR and CD70, is effective against EGFR and CD70 high expressed tumors

Abstract The epidermal growth factor receptor (EGFR) is highly expressed across a wide range of different cancers, and is a known driver of cancer growth; EGFR upregulation is frequently associated with adverse prognosis. Many patients undergoing EGFR-TKI (tyrosine kinase inhibitors) treatment will acquire resistance, which can be accompanied by increased expression of CD70 in some cancers. Simultaneous engagement of both EGFR and CD70 with a bispecific antibody (bsAb) is a potential strategy to overcome such resistance and achieve greater efficacy. To explore this strategy, we developed a novel IgG1 bsAb, BCG020, targeting EGFR and CD70. The parental EGFR antibody and CD70 antibody were developed from fully human common light chain antibody mice (RenLite®). When compared to EGFR benchmarks, the parental EGFR antibody exhibits similar affinity to both human and cynomolgus monkey EGFR, similar internalization activity in EGFR+ tumor cell lines, and superior blocking activity of EGF to EGFR in vitro. Furthermore, the EGFR parental antibody demonstrates good stability. The parental CD70 antibody exhibits high affinity to both human and monkey CD70, with superior internalization activity to benchmark antibody in CD70+ tumor cell lines, as well as superior blockade of CD70-induced signaling of CD27-Luc when compared to benchmark antibodies. When assembled into a bsAb, BCG020 exhitibs the same high affinity as both parental antibodies, with higher internalization activity than parental antibodies and benchmark antibodies. To evaluate the efficacy of BCG020 in vivo, we established cell line-derived xenograft (CDX) models in immunodeficient (B-NDG) mice. We observed that BCG020 treatment (10 mg/kg) potently inhibited growth of EGFR+CD70+ tumors. Taken together, these data indicate that BCG020 is a first-in-class bispecific antibody with potential to treat solid tumors with high expression of EGFR and CD70. Citation Format: Yongfei Yang, Xiaoxia Sun, Ziyan Li, Yue Yan, Linpeng Xu, Xiaoxue Wu, Luheng Du, Ying Zhu, Chaoshe Guo, W. Frank An. BCG020, a novel bispecific antibody targeting EGFR and CD70, is effective against EGFR and CD70 high expressed tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2724.