Development Of Antibody-drug Conjugates Research Articles

Combinatorial metabolic engineering of Streptomyces sp. CB03234-S for the enhanced production of anthraquinone-fused enediyne tiancimycins

BackgroundAnthraquinone-fused enediynes (AFEs) are excellent payloads for antibody-drug conjugates (ADCs). The yields of AFEs in the original bacterial hosts are extremely low. Multiple traditional methods had been adopted to enhance the production of the AFEs. Despite these efforts, the production titers of these compounds are still low, presenting a practical challenge for their development. Tiancimycins (TNMs) are a class of AFEs produced by Streptomyces sp. CB03234. One of their salient features is that they exhibit rapid and complete cell killing ability against various cancer cell lines.ResultsIn this study, a combinatorial metabolic engineering strategy guided by the CB03234-S genome and transcriptome was employed to improve the titers of TNMs. First, re-sequencing of CB03234-S (Ribosome engineered mutant strains) genome revealed the deletion of a 583-kb DNA fragment, accounting for about 7.5% of its genome. Second, by individual or combined inactivation of seven potential precursor competitive biosynthetic gene clusters (BGCs) in CB03234-S, a double-BGC inactivation mutant, S1009, was identified with an improved TNMs titer of 28.2 ± 0.8 mg/L. Third, overexpression of five essential biosynthetic genes, including two post-modification genes, and three self-resistance auxiliary genes, was also conducted, through which we discovered that mutants carrying the core genes, tnmE or tnmE10, exhibited enhanced TNMs production. The average TNMs yield reached 43.5 ± 2.4 mg/L in a 30-L fermenter, representing an approximately 360% increase over CB03234-S and the highest titer among all AFEs to date. Moreover, the resulting mutant produced TNM-W, a unique TNM derivative with a double bond instead of a common ethylene oxide moiety. Preliminary studies suggested that TNM-W was probably converted from TNM-A by both TnmE and TnmE10.ConclusionsBased on the genome and transcriptome analyses, we adopted a combined metabolic engineering strategy for precursor enrichment and biosynthetic pathway reorganization to construct a high-yield strain of TNMs based on CB03234-S. Our study establishes a solid basis for the clinical development of AFE-based ADCs.

Antibody-drug conjugates in solid tumors; new strategy for cancer therapy.

Antibody-drug conjugates (ADCs) have emerged as a novel class of anticancer treatment. ADCs are composed of three parts: a monoclonal antibody, a linker and a payload. A monoclonal antibody binds to the specific antigen present at the cancer cells, allowing selective delivery of the cytotoxic agents to the tumor site. Several ADCs are approved by the US Food and Drug Administration for the treatment of hematologic cancers and solid tumors with clinically meaningful survival benefit. However, the development of ADCs faces a lot of challenges and there is a need to get better understanding of ADCs in order to improve patient outcomes. Here, we briefly discuss the structure and mechanism of ADCs, as well as the clinical data of current approved ADCs in solid tumors.

Abstract PO3-03-05: Clinical outcomes in early-stage TNBC according to HER2-low status

Abstract Background: Clinical outcomes according to HER2 immunohistochemistry (IHC) in patients with early-stage HER2-negative breast cancer have differed across studies. For early-stage TNBC (eTNBC), less representation of this subtype in cohorts studied to date has limited interpretation of results. We sought to evaluate outcomes according to HER2 IHC in a multi-institutional cohort of patients (pts) with eTNBC who received neoadjuvant therapy (NAT). Methods: Pts diagnosed with stage I-III TNBC (including HR-low; ER and PR < 10%) who received NAT and underwent surgery between 1/1/16-6/30/19 were identified across three institutional prospective databases. HER2 was defined as low (1+ or 2+/ISH non-amplified) or HER2-0 according to local testing at diagnosis. Pathological complete response (pCR) was defined as no residual invasive disease in breast and axilla. Multivariable logistic regression was used to compare pCR rates (HER2-low vs HER2-0) adjusting for age at diagnosis, race, anatomic clinical stage, germline BRCA1/2 (gBRCA), histology, HR status and receipt of anthracycline and taxane NAT. Recurrence-free (RFS), distant recurrence-free (DRFS), and overall survival (OS) were estimated using the Kaplan-Meier method. Multivariable Cox proportional hazards model was used to estimate adjusted hazard ratios. Results: A total of 978 pts were identified of which 388 (39.7%) had HER2-low and 590 (60.3%) had HER2-0 tumors at diagnosis. Median age was 50.3 (range: 21.0-83.4) yrs. 174 (17.8%) pts had HR-low tumors. 142 (14.5%) pts had a known gBRCA mutation. 790 pts (80.8%) received anthracycline- and taxane NAT. No significant differences were observed in age, race, gBRCA, histology, HR status (low vs negative), clinical tumor size, or type of NAT between HER2-low and HER2-0 groups. Clinical nodal positivity was higher in HER2-low (55.2%) vs HER2-0 (46.6%), p=0.011. No significant difference in pCR was observed between HER2-low (32.0%) and HER2-0 (32.7%) groups, adjusted p=0.928. Among pts with residual disease (RD) post-NAT with HER2 IHC also available in the RD sample, 244/363 (67.2%) had concordant HER2 status. 70/225 (31.1%) of HER2-0 tumors at diagnosis had IHC expression post-NAT (66 HER2-low, 4 HER2-positive); 48/138 (34.8%) of HER2-low tumors at diagnosis were HER2-0 post-NAT. At a median follow-up of 3.1 yrs, RFS did not significantly differ between HER2-low vs HER2-0 pts with pCR (p=0.368) or in those with RD post-NAT (p=0.573). Similarly, DRFS did not differ according to HER2 category for pts with pCR (p=0.509) or RD (p=0.812), nor did OS for pts with pCR (p=0.514) or RD (p=0.285) (Table 1). Conclusion: In a large cohort of pts with eTNBC treated with NAT, HER2-low status was not associated with pCR or survival after adjusting for clinical factors. High rate of discordance in HER2 IHC between diagnostic and RD post-NAT was observed, highlighting the importance of repeat IHC testing in serial samples given the development of antibody drug conjugates for HER2-low breast cancer. Table 1. RFS, DRFS and OS by HER2 expression Citation Format: Ana Garrido-Castro, Danielle Brandes Zakon, Qingchun Jin, Michael Grimm, Akshara Singareeka Raghavendra, Melissa Hughes, Mathew Cherian, Julie Vincuilla, Tonia Parker, Paolo Tarantino, Elizabeth Mittendorf, Tari King, Vicente Valero, Debu Tripathy, Bhuvaneswari Ramaswamy, Sara Tolaney, Nabihah Tayob, Nancy Lin, Daniel Stover, Carlos Barcenas. Clinical outcomes in early-stage TNBC according to HER2-low status [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-03-05.

Abstract PO4-06-02: Frequency of presentation and outcomes of stage IV breast cancer over the past decade: a population-based study

Abstract Background: Over the past decade, there have been major advances for the treatment of patients (pts) with metastatic breast cancer (MBC). For pts with hormone receptor (HR) positive, human epidermal growth factor receptor 2 (HER2) negative MBC, newer treatments include CDK 4/6 inhibitors among other targeted therapies; for HER2+ MBC, we have seen the development of antibody-drug conjugates (ADC) and other anti-HER2 agents; and for triple-negative breast cancer (TNBC) options include immunotherapy, ADCs, and PARP inhibitors. The aims of this study were to evaluate time trends in clinical characteristics, frequency of presentation and outcomes of pts with stage IV breast cancer over the past decade. Methods: Using data from the Surveillance, Epidemiology, and End Results (SEER) program, we identified pts diagnosed with breast cancer between 2010-2020. Pts required to have known HR and HER2 status and known stage at diagnosis. We described the proportion of stage IV disease overall for each tumor subtype and evaluated trends in the proportion of stage IV over time. Outcomes were evaluated by breast cancer-specific survival (BCSS) using Kaplan-Meier. Among stage IV pts, we conducted multivariable cox models to assess changes in BCSS over time, stratified by tumor subtype and adjusted for age at diagnosis, race and ethnicity, sex, tumor grade, histology, surgery, radiation therapy, chemotherapy, sites of distant metastases, marital status, median household income, rurality and US region. We evaluated overall survival as a secondary endpoint. Results: We included 443,631 pts of whom 24,788 (5.59%) had stage IV disease at diagnosis. Pts with stage IV breast cancer had a median age of 61 years and a median follow up of 57 months (IQR 28 – 89 months). When compared to stage I-III, stage IV pts were more often of Black race (10.4% for stage I-III vs 15.7% for stage IV), less often married (56.7% vs 43.5%, respectively) and less often HR+/HER2- (74.1% vs 59.9%, respectively); all p < 0.001. Among pts with stage IV, the frequency of each metastatic site at initial diagnosis was: bone 66.45%, lung 30.47%, liver 25.79%, and brain 7.9%. Across all years (2010-2020), the proportion of stage IV disease varied significantly by tumor subtype: 4.6% in HR+/HER2-, 8.5% in HR+/HER2+, 11.3% in HR-/HER2+, and 7.1% in TNBC; p < 0.001. The proportion of stage IV increased modestly during 2010-2020 in HR+/HER2- tumors (4.6% in 2010 to 5.1% in 2020, p for trend = 0.01), in HR-/HER2+ (10% in 2010 to 13.7% in 2020, p for trend < 0.001), and in TNBC (6.9% in 2010 to 7.8% in 2020, p for trend = 0.001). No significant difference was observed in the proportion of stage IV over time in HR+/HER2+ (p for trend = 0.76). Rate of BCSS at 5 years was: 32.51% for HR+/HER2-, 43.92% for HR+/HER2+, 37.28% for HR-/HER2+, and 10.97% for TNBC; p < 0.001. In adjusted Cox models, BCSS improved significantly over time for each additional year in HR+/HER2- (adjusted Hazard Ratio [HzR] 0.982, p = 0.01), in HR+/HER2+ (adjusted HzR 0.945, p < 0.001), and in HR-/HER2+ (adjusted HzR 0.959, p = 0.03). The improvement in BCSS over time in TNBC was not significant (adjusted HzR 0.982, p = 0.15). Overall survival results were similar to those of BCSS both in 5-year rates as well as Cox model results. Conclusions: In this population-based study, we saw that the proportion of pts presenting with de-novo stage IV varied significantly by tumor subtype, being as low as 4.6% in HR+/HER2- and as high as 11.3% in HR-/HER2+ disease. These proportions have unfortunately increased over time in most subtypes, highlighting a need for additional research to identify the reasons behind these trends. BCSS improved significantly over time in HR+/HER2- and in both HER2+ subtypes, coinciding with a time period of important therapeutic advances. TNBC remains a highly unmet medical need and further follow up is needed to assess the impact of newer treatments. Citation Format: Jorge Avila, Julieta Leone, Nabihah Tayob, Carlos Vallejo, Michael Hassett, Nancy Lin, Sara Tolaney, Jose Leone. Frequency of presentation and outcomes of stage IV breast cancer over the past decade: a population-based study [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-06-02.

Abstract PO1-13-07: LIV-1 and Trop2 expression using Immunohistochemistry as prognostic markers in early breast cancer

Abstract Backgrounds: Antibody-drug conjugate (ADC) has emerged as treatment option for breast cancer (BC). The ASCENT and TROPiCS-02 trial of Sacituzumab Govitecan, a Trop2 ADC, gained great success in TNBC and HR+ BC. Other targets, such as LIV-1, are being investigated for TNBC and HR+ BC. The clinical trials of Ladiratuzumab, an ADC of LIV-1 Ab and MMAE, are ongoing. However, unlike Her2 protein, the predictive and prognostic role of LIV-1 and Trop-2 has not been fully investigated. Therefore, we aimed to analyze the relationship between expression level and clinicopathological features and explore the value of the markers among subtypes of BC. Methods: TMA were selected from 1,349 breast cancer specimen of patients who received curative surgery from 2008 to 2012 at Seoul National University Hospital and IHC staining was performed on the TMA using LIV-1 antibody (HPA042377 manufactured by Sigma-Aldrich, St. Louis, MO, dilution ratio of 1:300) and Trop2 antibody(HPA055067, dilution ratio of 1:75). All IHC slides were carefully evaluated by trained pathologists. IHC expression was assessed as intensity (negative:0, weak:1, moderate:2, and strong:3). A modified histochemical score(H-score) was calculated from the intensity multiplied by the percentage of positive cells. Results: A total of 1119 patients from selected samples were included. The median age was 49 (range 25-90). Most patients had early T stage disease (n=513, 569, 29, 7, for pT1, pT2, pT3, pT4, respectively). The percentage of nodal metastasis (N=687 vs 432 in pN0 vs ≥pN1, 61.4 vs 38.6%) and LVI (N= 678 vs 441 in neg vs pos, 60.6% vs 38.6%). 713 pts (63.7%) were HR+ subtype, 200(17.9%) of Her2+ and 206(18.4%) of TNBC. We divided patients into negative/low/high expression groups according to LIV-1 expression by median expression level (cutoff H-score = 100): 479 of 1119 (42.8%) LIV-1-negative; 270 (42.2%) LIV-1-low expression and 370 (57.8%) LIV-1-high. Patients with LIV-1-low or -high tumors showed better DFS (156.2 vs 158.8 vs 159.95 mo for LIV-1-negative, -low, -high, respectively; P=0.0072) and OS(157.6 vs 163 vs 165.4 mo, P=0.0029) . HR+ subgroup, (N= 193 vs 228 vs 354 for neg vs low vs high), followed overall tendency (153.4vs 158.3 vs 159.9mo, P=0.0571 in DFS, 155.6vs160.2 vs165.4mo, P=0.0627 in OS, respectively) In contrast, for patients with TNBC, LIV-1-high expression showed the worst DFS and OS. (N = 183 vs 18 vs 5 in neg vs low vs high group, 159.7 vs 160.7 vs 93.1mo, P value=0.1854 for DFS, 160.8 vs 160.7 vs 93.1 mo, P=0.0334 for OS, respectively) LIV-1 expression failed to prove a prognostic value in Her2+ BC. We also analyzed Trop2 expression by dividing patients into low/intermediate/high by the method used previously in ASCENT trial. 1009 of 1119 (n=341 in low, 363 in intermediate, 305 in high expression) patients had Trop2 expression. There was no statistically significant difference in DFS and OS among all subtypes. In multivariate analysis, neither LIV-1 or Trop-2 had a prognostic role. Conclusion: The analysis of clinicopathological findings of LIV-1 protein indicates their values for prognosis markers, especially in HR+ BC and TNBC. Trop2 expression has no prognostic role in line with previous research. Further studies are warranted to explore targetable biomarkers for the development of appropriate ADCs. Citation Format: Yeonjoo Choi, Han Suk Ryu, Jiwon Koh, Dae-Won Lee, Kyung-hun Lee, Seock-Ah Im. LIV-1 and Trop2 expression using Immunohistochemistry as prognostic markers in early breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-13-07.

Development and therapeutic evaluation of 5D3(CC-MLN8237)3.2 antibody-theranostic conjugates for PSMA-positive prostate cancer therapy.

Prostate cancer (PC) is an aggressive cancer that can progress rapidly and eventually become castrate-resistant prostate cancer (CRPC). Stage IV metastatic castrate-resistant prostate cancer (mCRPC) is an incurable late-stage cancer type with a low 5-year overall survival rate. Targeted therapeutics such as antibody-drug conjugates (ADCs) based on high-affinity monoclonal antibodies and potent drugs conjugated via smart linkers are being developed for PC management. Conjugating further with in vitro or in vivo imaging agents, ADCs can be used as antibody-theranostic conjugates (ATCs) for diagnostic and image-guided drug delivery. In this study, we have developed a novel ATC for PSMA (+) PC therapy utilizing (a) anti-PSMA 5D3 mAb, (b) Aurora A kinase inhibitor, MLN8237, and (c) for the first time using tetrazine (Tz) and trans-cyclooctene (TCO) click chemistry-based conjugation linker (CC linker) in ADC development. The resulting 5D3(CC-MLN8237)3.2 was labeled with suitable fluorophores for in vitro and in vivo imaging. The products were characterized by SDS-PAGE, MALDI-TOF, and DLS and evaluated in vitro by optical imaging, flow cytometry, and WST-8 assay for cytotoxicity in PSMA (+/-) cells. Therapeutic efficacy was determined in human PC xenograft mouse models following a designed treatment schedule. After the treatment study animals were euthanized, and toxicological studies, complete blood count (CBC), blood clinical chemistry analysis, and H&E staining of vital organs were conducted to determine side effects and systemic toxicities. The IC50 values of 5D3(CC-MLN8237)3.2-AF488 in PSMA (+) PC3-PIP and PMSA (-) PC3-Flu cells are 8.17nM and 161.9nM, respectively. Pure MLN8237 shows 736.9nM and 873.4nM IC50 values for PC3-PIP and PC3-Flu cells, respectively. In vivo study in human xenograft mouse models confirmed high therapeutic efficacy of 5D3(CC-MLN8237)3.2-CF750 with significant control of PSMA (+) tumor growth with minimal systemic toxicity in the treated group compared to PSMA (-) treated and untreated groups. Approximately 70% of PSMA (+) PC3-PIP tumors did not exceed the threshold of the tumor size in the surrogate Kaplan-Meyer analysis. The novel ATC successfully controlled the growth of PSMA (+) tumors in preclinical settings with minimal systemic toxicities. The therapeutic efficacy and favorable safety profile of novel 5D3(CC-MLN8237)3.2 ATC demonstrates their potential use as a theranostic against aggressive PC.

Monomethyl Auristatin E (MMAE), a Payload for Multiple Antibody Drug Conjugates (ADCs), Demonstrates Differential Red Blood Cell Partitioning Across Human and Animal Species

Background: Monomethyl auristatin E (MMAE) has been used as a payload for several Food and Drug Administration (FDA) approved antibody-drug conjugates (ADCs). It is known that MMAE is released from the ADC following binding, internalization and proteolytic degradation in target tissues. A striking discrepancy in systemic MMAE levels has been observed across species with 50-fold higher MMAE levels in human than that in rodents when normalized by ADC dose with unknown mechanism. Hypothesis and purpose: Multiple factors could affect systemic MMAE levels such as production and elimination of unconjugated MMAE following ADC dosing. In this study, we have explored whether MMAE displays differential red blood cell (RBC) partitioning across species that may contribute to the different MMAE levels seen between human and animals. Experiments: To determine MMAE RBC partitioning, tritium labeled MMAE ([3H]-MMAE) was incubated in whole blood from mice, rats, monkeys and humans in vitro, then RBC partitioning was determined and compared across species. To test whether MMAE released from the ADC would show any difference in RBC partitioning, pinatuzumab vedotin or polatuzumab vedotin was administered to mice, rats, and monkeys. MMAE levels were measured in both blood and plasma, and the ratios of MMAE levels were calculated as blood-to-plasma ratio (in vivo RBC partitioning). Results: Our in vitro data showed that unconjugated MMAE has a species-dependent RBC partitioning with strong RBC partitioning in mouse, rat, followed by monkey blood, whereas minimal RBC partitioning was seen in human blood. Incubation of 2 nM of MMAE in mouse blood resulted in a blood-to-plasma ratio of 11.8 ± 0.291, followed by rat, monkey, and human at 2.36 ± 0.0825, 1.57 ± 0.0250, and 0.976 ± 0.0620, respectively. MMAE RBC partitioning is also concentration-dependent, with an inverse relationship between RBC partitioning and MMAE concentration (higher RBC partitioning at lower concentration). In vivo dosing of pinatuzumab vedotin in mouse displayed systemic MMAE at about a 5-fold higher blood concentration compared to plasma concentration once MMAE reached a pseudo-equilibrium, while systemic MMAE from blood and plasma concentration showed a 1.65-fold difference in rat. Implication and conclusion: These data demonstrated that MMAE has a distinct RBC partitioning across different species, which may contribute to, at least in part, to the differential in the systemic MMAE levels observed in vivo between preclinical and clinical studies. These findings highlight the importance of fully characterizing the ADME properties of both the ADC and its payload, to enable better translation from animals to human for ADC development.

Antibody-Drug Conjugates in the Treatment of Genitourinary Cancers: An Updated Review of Data.

The treatment landscape of genitourinary cancers has significantly evolved over the past few years. Renal cell carcinoma, bladder cancer, and prostate cancer are the most common genitourinary malignancies. Recent advancements have produced new targeted therapies, particularly antibody-drug conjugates (ADCs), due to a better understanding of the underlying oncogenic factors and molecular mechanisms involved. ADCs function as a 'drug delivery into the tumor' system. They are composed of an antigen-directed antibody linked to a cytotoxic drug that releases cytotoxic components after binding to the tumor cell's surface antigen. ADCs have been proven to be extremely promising in the treatment of several cancer types. For GU cancers, this novel treatment has only benefited patients with metastatic urothelial cancer (mUC). The rest of the GU cancer paradigm does not have any FDA-approved ADC treatment options available yet. In this study, we have thoroughly completed a narrative review of the current literature and summarized preclinical studies and clinical trials that evaluated the utility, activity, and toxicity of ADCs in GU cancers, the prospects of ADC development, and the ongoing clinical trials. Prospective clinical trials, retrospective studies, case reports, and scoping reviews were included.

Precision Medicine in Rheumatic Diseases: Unlocking the Potential of Antibody-Drug Conjugates.

In the era of precision medicine, Antibody-Drug Conjugates (ADCs) have emerged as a cutting-edge therapeutic strategy. These innovative compounds combine the precision of monoclonal antibodies with the potent cell-killing or immune-modulating abilities of attached drug payloads. This unique strategy not only reduces off-target toxicity but also enhances the therapeutic effectiveness of drugs. Beyond their well-established role in oncology, ADCs are now showing promising potential in addressing the unmet needs in the therapeutics of rheumatic diseases. Rheumatic diseases, a diverse group of chronic autoimmune diseases with varying etiologies, clinical presentations, and prognoses, often demand prolonged pharmacological interventions, creating a pressing need for novel, efficient and low-risk treatment options. ADCs, with their ability to precisely target the immune components, have emerged as a novel therapeutic strategy in this context. This review will provide an overview of the core components and mechanisms behind ADCs, a summary of the latest clinical trials of ADCs for the treatment if rheumatic diseases, as well as a discussion on the challenges and future prospects faced by the development of next-generation ADCs. Significance Statement There is a lack of efficient and low-risk targeted therapeutics for rheumatic diseases. Antibody-drug conjugates, a class of cutting-edge therapeutic drugs, have emerged as a promising targeted therapeutic strategy for rheumatic disease. While there is limited literature summarizing the progress of antibody-drug conjugates in the field of rheumatic disease, updating the advancements in this area provides novel insights into the development of novel anti-rheumatic drugs.

Radiotherapy activates picolinium prodrugs in tumours.

Radiotherapy-induced prodrug activation provides an ideal solution to reduce the systemic toxicity of chemotherapy in cancer therapy, but the scope of the radiation-activated protecting groups is limited. Here we present that the well-established photoinduced electron transfer chemistry may pave the way for developing versatile radiation-removable protecting groups. Using a functional reporter assay, N-alkyl-4-picolinium (NAP) was identified as a caging group that efficiently responds to radiation by releasing a client molecule. When evaluated in a competition experiment, the NAP moiety is more efficient than other radiation-removable protecting groups discovered so far. Leveraging this property, we developed a NAP-derived carbamate linker that releases fluorophores and toxins on radiation, which we incorporated into antibody-drug conjugates (ADCs). These designed ADCs were active in living cells and tumour-bearing mice, highlighting the potential to use such a radiation-removable protecting group for the development of next-generation ADCs with improved stability and therapeutic effects.

Abstract 2615: Preclinical development of a next generation antibody drug conjugate (ADC) targeting cMET for treatment of solid tumors

Abstract cMET (mesenchymal-epithelial transition factor), which belongs to the MET family, is a type of receptor tyrosine kinase that is expressed on the surfaces of various epithelial cells. The elevation of cMET can promote the development and progression of multiple cancers. cMET- ADCs have shown promising clinical activity with highest cMET levels, indicating tumor cMET levels may be limited for efficacy. YL211, a novel cMET-ADC was developed by leveraging on MediLink’s tumor microenvironment activable linker-payload platform (TMALIN platform), which could release the payload both in the tumor cell and in the tumor microenvironment. YL211 is comprised of an anti-cMET humanized monoclonal antibody conjugated to a novel topoisomerase 1 inhibitor via a protease-cleavable linker. The novel linker-payload and site-specific conjugation of YL211 results in a homogeneous product with a DAR (Drug-to-Antibody Ratio) of 8. Furthermore, the advancement in linker-payload design has facilitated the development of more hydrophilic ADCs, which exhibit minimal impact on the antibody's characteristics while being less prone to aggregation and showcasing lower systematic clearance. Along with release of the payload in the tumor microenvironment extracellularly, upon binding to cMET on tumor cell surfaces, the antigen-ADC complexes are internalized to lysosomal vesicles and subsequently release the toxic payload. The cytotoxic features drive cell cycle arrest, apoptosis, elevation of PARP/caspase 7 expression, as well as cell death and bystander effect. These findings translated in vivo where YL211 showed superior efficacy in causing tumor regressions and good tolerability in both cell lines and patient-derived xenograft (CDX and PDX) mouse models with both low to high cMET expression levels (H358, CR5088, ect.). The in vivo pharmacokinetics in monkeys showed that YL211 is highly stable with less than 0.1% (molar ratio) payload released in circulation. GLP safety evaluations demonstrated good safety profiles and no off-target toxicity in monkeys. These non-clinical data suggest the stability of the linker in circulation as well as efficient release of the payload in tumors with improved therapeutic window. Taken together, preclinical data suggest that YL211 could be a promising treatment strategy for cMET positive cancer patients. Citation Format: Liang Xiao, Wei Lian, Shuai Song, Qigang Liu, Qing Zong, Sasha Stann, Jiaqiang Cai, Tongtong Xue. Preclinical development of a next generation antibody drug conjugate (ADC) targeting cMET for treatment of 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 2615.

Abstract 739: AMT-253, a first-in-class MUC18-targeting antibody-drug conjugate, for the treatment of MUC18-positive solid tumors

Abstract MUC18, also known as MCAM (melanoma cell adhesion molecule) or CD146, is a type I transmembrane glycoprotein originally identified as a cell adhesion molecule of melanoma that plays important roles in tumor growth and progression including tumor angiogenesis, epithelial-mesenchymal transition (EMT), and metastasis. MUC18 is overexpressed in a variety of solid tumors including (but not limited to) melanoma, osteosarcoma, head and neck, lung, esophagus, breast, ovarian, and cervical cancer and hepatocellular carcinoma, with restricted presence on normal healthy tissues, mainly on blood vessels and a minor subset of T helper cells. Limited expression in normal tissues and high expression in tumors makes MUC18 a promising target for the development of antibody drug conjugate (ADC). AMT-253 is a novel ADC targeting MUC18. It is composed of a humanized MUC18-targeting IgG1 antibody conjugated with Exatecan, a topoisomerase I inhibitor, attached to a proprietary self immolative T moiety linker through reduced interchain disulfide bonds with a drug-to-antibody ratio (DAR) of 8. AMT-253 was stable in vitro with less than 0.5% of payload releasing when incubated with human, monkey, mouse or rat plasma for 21 days at 37°C. AMT-253 selectively bound to cell surface MUC18 and was efficiently internalized into MUC18-positive cancer cells. AMT-253 exhibited antiproliferative activity against several MUC18-positive cancer cell lines and demonstrated potent bystander killing effect in vitro. Treatment with AMT-253 resulted in tumor growth inhibition or regression in a panel of MUC18 positive cell line derived xenograft (CDX) or patient derived xenograft (PDX) models. In addition, AMT-253 was well tolerated in a GLP compliant toxicity study in cynomolgus monkeys at dose levels ≤45 mg/kg. Taken together, our preclinical in vivo efficacy and toxicity studies demonstrated that AMT-253 has a wide therapeutic window and could potentially provide benefits for cancer patients with highly unmet medical needs. AMT-253 is currently being evaluated in a Phase I first-in-human clinical trial (NCT05906862). Citation Format: Jing Shi, Jiaqing Yan, Yaling Huang, Jun Guo, Yan Kong, Xun Meng, Shu-Hui Liu. AMT-253, a first-in-class MUC18-targeting antibody-drug conjugate, for the treatment of MUC18-positive 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 739.

Abstract 2618: BCG017, a bispecific ADC targeting PTK7 and EGFR exhibits anti-tumor efficacy in PDX models

Abstract EGFR is a favored target in various cancer therapies. However, EGFR-based treatments have been challenged by drug resistance and cytotoxicity. Targeting EGFR with an antibody-drug conjugate (ADC) is a promising new therapeutic strategy to address the drug resistance, due to the potent killing effects of the payload. Furthermore, we hypothesized that development of a bispecific ADC (bsADC) targeting EGFR and another tumor-associated antigen (TAA) might improve the tumor selectivity, thereby limiting the on-target off-tumor toxicity. One such TAA candidate is PTK7, which belongs to the same receptor tyrosine kinase family as EGFR, and is co-expressed with EGFR in multiple types of solid tumors, including lung, esophageal, head and neck, and colon cancers. Herein, we developed a fully human anti-human PTK7 EGFR bsAb using our proprietary common light chain RenLite® mouse platform and knobs-into-holes technology. The anti-PTK7 EGFR bsAb demonstrates good purity by SEC-HPLC, and can cross-react with human and cynomolgus monkey antigens with favorable affinity. The PTK7 x EGFR bsAb demonstrated higher binding to various types of cancer cell lines, as well as enhanced internalization in vitro compared with PTK7 parental antibody. Next, the bsAb was conjugated with monomethyl auristatin E (MMAE) to generate an anti-PTK7 x EGFR bsADC, BCG017, with a drug-to-antibody ratio of approximately 4. BCG017 demonstrated superior anti-tumor activity compared to benchmarks and its PTK7 parental ADC in cell-derived non-small-cell lung cancer (NSCLC) xenografts, and showed enhanced anti-tumoractivity when compared to benchmark ADCs and parental ADCs in patient-derived breast and pancreatic ductal adenocarcinoma xenograft models. These results suggest that BCG017 have the potential to be a novel therapeutic alternative for PTK7 and EGFR co-expressing tumors. Citation Format: Sufei Yao, Chengzhang Shang, Zhuolin Li, Gao An, Chaoshe Guo, W. Frank An, Yi Yang. BCG017, a bispecific ADC targeting PTK7 and EGFR exhibits anti-tumor efficacy in PDX 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 2618.

Abstract 4689: 4HF CancerDataMiner platform accelerates target discovery and evaluation

Abstract For the management of metastasized cancer, tumor-targeted chemotherapies with improved therapeutic index are urgently needed. Over the last 25 years, antibody drug conjugates (ADCs) targeting cancer-associated proteins have emerged as a promising new treatment modality. While the first ADCs were registered for the treatment of acute leukemias and lymphomas, more recently at least six ADCs were approved by the FDA for solid tumors. Despite these successes, clinical development of ADCs remains a risky exercise as evident from the failure of more than 20 pivotal clinical phase III trials. A narrow therapeutic index due to excessive toxicity of potent payloads, little therapeutic activity, and difficulties in chemistry, manufacturing, and controls were among the limiting factors. Meanwhile, ADC technology and production have advanced substantially. More than 100 ADCs are currently in clinical development in approximately 600 clinical trials, with some of the investigated ADCs being close to approval. While issues related to ADC linkers and payloads have been extensively investigated and many problems have been solved, the choice of molecular targets for ADCs remains challenging. This is, in part, due to a lack of information about candidate targets: while knowledge about generally accepted cancer targets such as ERBB2 is abundant, data about many novel targets is still scarce. In particular, reliable information about expression in normal tissues, subcellular localization and protein function is often missing. 4HF Biotec has established the CancerDataMiner, an in-silico platform dedicated to cancer data mining by integrating large OMICS datasets that are connected and aligned. It allows molecular analyses of all human genes in >12,000 tumor samples and >14,000 normal tissues, using proprietary tools and widgets for visualization and statistics. Target evaluations are performed at the DNA, RNA and protein levels and employ complementary tools such as pathway analysis and literature search to obtain a comprehensive picture of candidate targets. Using this approach, we recently reviewed the molecular targets of all ADCs that either have already received approval or are in clinical development. As proof-of-concept for the CancerDataMiner, here we analyze targets of ADCs and compare our findings with the clinical data obtained for those ADCs. The profiles of selected ADC targets will be discussed, among them CD79B and CD33 for hematologic malignancies and FOLR1 and Claudin 18.2 for solid cancers. We will demonstrate that proteins with very diverse profiles can make good ADC targets. In conclusion, the 4HF CancerDataMiner is a platform which has proven its value for the identification and characterization of cancer-associated targets for ADC development for all tumor types. Citation Format: Heinz-Herbert Fiebig, Anne-Lise Peille, Mirko Schmitz, Vincent Vuaroqueaux, Thomas Metz. 4HF CancerDataMiner platform accelerates target discovery and 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 4689.

Abstract 513: Intra-tubular damage is targeted by maytansinoids and rescued by NF1: Revisiting mechanism and biomarkers of an established ADC payload

Abstract There is great interest in the identification of biomarkers to guide development of antibody-drug conjugates (ADC). We previously showed that loss of Neurofibromatosis 1 (NF1), a gene frequently mutated across cancers, enhances the activity of DM1, the maytansinoid payload of T-DM1, through a novel function in regulating microtubule (MT) dynamics. Maytansinoids are puzzlingly more effective in cells (in the nanomolar range) vs in vitro (in the micromolar range). Since maytansinoids bind at the interface between tubulin dimers, they are thought to only bind soluble tubulin dimers or MT ends, which would suggest very few binding sites available for pharmacological interaction in vivo, at odds with data. Here we investigated the interaction of DM1 with NF1 and MTs, using cellular and reductionist in vitro systems. To measure in vivo MT dynamics, we transiently transfected the MT end-binding protein EB3-GFP and reconstructed MT trajectories by live-cell imaging. Upon DM1 treatment, KO cells showed a highly significant reduction in MT speed, demonstrating a direct role for NF1 on MT dynamics in cells. In turbidity-based tubulin polymerization assays, recombinant NF1 greatly accelerated polymerization, and completely rescued DM1-induced inhibition. Visual inspection of fluorescent MTs showed that NF1 induced significant MT bundling, a defining feature of many MT-associated proteins, which generates signal indistinguishable from true MT polymerization in turbidity assays. To follow the dynamics of individual microtubules, we applied Total Internal Reflection (TIRF) microscopy on glass-immobilized MTs. As expected, polymerization in the presence of NF1 led to a significant increase in MT dynamics (elongation speed, rescue and catastrophe rate). Expectedly, DM1 led to significant reduction in the fraction of elongating MTs and speed, but these defects were completely or partially rescued by NF1. Importantly, DM1 did not only lead to MT shortening (as proposed by the current model), but also to clear and frequent MT fracturing, indicating that the drug is not only engaging MT ends but also intra-tubular binding sites. This is consistent with recent models of MT formation which incorporate the frequent presence of areas of discontinuity or damage induced by mechanical stress, exposing intra-tubular DM1 binding sites. Interestingly, adding NF1 to DM1-treated MTs generated areas of de novo intra-tubular tubulin insertion, coincident with damaged sites, suggesting an entirely novel role for NF1 in MT repair.In conclusion, we provide evidence for a model in which maytansinoids bind not only to soluble tubulin dimers and MT ends, but also to intra-tubular damaged sites. Thus, the number of binding sites in cells would be proportional to MT damage, suggesting a mechanism for differential efficacy across tumor types and a potential avenue for combinatorial drug development. Citation Format: Eleonora Messuti, Bruno Achutti Duso, Alessia Castiglioni, Giulia Tini, Emanuele Bonetti, Giuseppe Ciossani, Silvia Monzani, Daria Khuntsariya, Zdeněk Lánský, Marcus Braun, Luigi Scietti, Luca Mazzarella. Intra-tubular damage is targeted by maytansinoids and rescued by NF1: Revisiting mechanism and biomarkers of an established ADC payload [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 513.

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 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 4708: Potency and selectivity of ERBB2-targeting antibody drug conjugates in vitro

Abstract Previously we have reported on the relevance of the joint ProLiFiler and Cancer DataMiner platforms when studying the antitumor efficacy of novel antibody-drug conjugates (ADCs), such as Sacituzumab govitecan. In this study, we use our platforms to investigate and compare the selectivity of two FDA-approved Trastuzumab-based ADCs targeting ERBB2: Kadcyla, the first-in-class ADC employing a microtubule inhibitor bound via a non-cleavable linker, and Enhertu, which combines a topoisomerase inhibitor with a protease-cleavable linker. We performed a cell proliferation and survival assay using the ProLiFilerTM (Reaction Biology) to characterize the in vitro antitumor effects of Enhertu, Kadcyla, and their individual cytotoxins, deruxtecan & mertansine on 160 human cancer cell lines (CLs). The resulting data were uploaded to 4HF´s Cancer DataMiner platform for data analysis. First, we confirmed high potency and limited selectivity of mertansine, as it displayed an IC50 < 250 nM (mean: 27.3 nM) in 95% of cell lines (CLs). A COMPARE analysis confirmed that mertansine data correlated best with those of other microtubule inhibitors in our drug databases (e.g., MMAE, Spearman rho = 0.79, p=9.6E-27). Conversely, a paired analysis showed that CLs were less sensitive to Kadcyla than to mertansine except for CLs overexpressing ERBB2. The 160-CL panel included a total of nine CLs (six mammary, two gastric, one ovarian) with strong ERBB2 overexpression (Affymetrix data >10), mostly associated with ERBB2 gene amplification; seven of them were the most sensitive CLs to Kadcyla (IC50 < 6 nM). Focusing solely on breast cancer cell lines (breast cancer is the approved indication for Kadcyla), we observed a strong correlation between sensitivity to Kadcyla and ERBB2 expression (Spearman r = -0.78, p = 0.003). Interestingly, in line with published data, the ERBB2-overexpressing cell lines SNU-216 (stomach cancer) and JIM-T1 (breast cancer) were less sensitive to Kadcyla. In non-ERBB2-amplified CLs, Kadcyla sensitivity did not correlate with ERBB2 expression levels. Focusing on ERBB2-negative CLs such as hematological CLs, we unexpectedly observed consistent cell inhibition (mean IC50 =33 nM) with Kadcyla, suggesting that the ADC was cleaved, and the payload released into the medium. Overall, this study has demonstrated the relevance of our platforms to study and compare newly developed ADCs. Cytotoxic activities recorded for conjugates and free payloads, along with molecular data for the test cell lines, provides insight in the mode of action of conjugates and their target cell populations and hence can support the development of ADCs. Investigations are underway with Enhertu and its payload deruxtecan, and results will be compared with those obtained for Kadcyla and mertansine. Citation Format: Anne-Lise Peille, Kaja Holtorf, Pablo Anton Garcia, Nadine Obier, Daniel Feger, Thomas Metz, Sebastian Dempe, Heinz-Herbert Fiebig, Jan Ehlert, Vincent Vuaroqueaux. Potency and selectivity of ERBB2-targeting antibody drug conjugates in vitro [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 4708.

Abstract 2610: Discovery of AM E3: A novel α-TLSPR monoclonal antibody for the ADC treatment of CRLF2-rearranged Ph-like B-ALL

Abstract Background: Patients affected by Ph-like B-cell acute lymphoblastic leukemia (B-ALL) have a high mortality rate and elevated levels of disease relapse upon chemotherapy treatment. More than 60% of Ph-like B-ALL is characterized by genomic rearrangements resulting in high expression level of the CRLF2/TLSPR receptor on the cell surface. While significant advances have been made in developing targeted agents for B-ALL treatment, the CRLF2-rearranged subpopulation is in complete absence of selective and effective therapies. Here, we report the identification of α-TLSPR monoclonal antibodies with therapeutic potential and the preclinical validation of AM E3 as an Antibody-Drug Conjugate (ADC) for the treatment of CRLF2-rearranged Ph-like B-ALL. Material and Methods: Leveraging a high-complex phage display platform we have first identified several fully human α-TLSPR monoclonal antibodies with single-digit nanomolar affinity for the target. Among them, AM E3 emerged as suitable for ADC development due to its rapid internalization by CRLF2-expressing cells. As a proof of concept, upon conjugation with the cytotoxic payload tesirine (SG3249) AM E3 was validated for anti-tumor response in both CRLF2-rearranged in vitro cell lines and in vivo xenograft models. Further, since human/mouse CRLF2 homology is low (˂35%), we have also developed an ADC surrogate against murine TLSPR to establish a therapeutic window for the ADC therapy in CRLF2-rearranged preclinical models. Results: ADC AM E3 demonstrated strong cytotoxicity (EC50=40pM) on cell lines derived from hCRLF2-rearranged B-ALL patients. In xenograft models a single injection of ADC AM E3 (0.7mg/Kg) showed antitumoral activity proportional to hCRLF2/TSLPR receptor density at the cell surface. Moreover, ADC AM E3 treatment reduces tumor growth and disease burden of hCRLF2-rearranged B-ALL orthotopic xenografts and extends survival over standard induction therapy. ADC surrogate mB4 AM23, which retains similar properties as the human ADC AM E3, reduces tumor growth and is well tolerated in xenograft models engineered to express mCRLF2/TSLPR. Conclusions: AM E3 is a novel α-TLSPR mAb with ideal features for the development of a first-in-class ADC therapy. α-TLSPR ADC demonstrated in vitro and in vivo efficacy as a monotherapy for the treatment of CRLF2/TLSPR rearranged Ph-like B-ALLs and proven to be well-tolerated in surrogate animal studies. Citation Format: Claudia Dall'Armi, Valentina Di Biasio, Chantal Paolini, Daniela Natale, Francesco Scalabrì, Antonino Missineo, Nadine Alaimo, Maurizio Nuzzo, Anna Demartis, Pamela Di Pasquale, Roberto Benoni, Fulvia Caretti, Elisa Beghetto, Fabrizio Colaceci, Romina Sasso, Vincenzo Pucci, Alessandro Carugo, Licia Tomei, Carlo Toniatti. Discovery of AM E3: A novel α-TLSPR monoclonal antibody for the ADC treatment of CRLF2-rearranged Ph-like B-ALL [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 2610.

Combinatorial Biosynthesis of 3-O-Carbamoylmaytansinol by Rational Engineering of the Tailoring Steps of Ansamitocins.

Currently, most maytansine-containing antibody-drug conjugates (ADCs) in clinical trials are prepared with DM1 or DM4, which in turn is synthesized mainly from ansamitocin P-3 (AP-3), a bacterial maytansinoid, isolated from Actinosynnema pretiosum. However, due to the high self-toxicity of AP-3 to A.pretiosum, the yield of AP-3 has been difficult to improve. Herein, a new maytansinoid with much lower self-toxicity to A.pretiosum, 3-O-carbamoylmaytansinol (CAM, 3), was designed and generated by introducing the 3-O-carbamoyltransferase gene asc21b together with the N-methyltransferase genes from exogenous maytansinoid gene clusters into the 3-O-acyltransferase gene (asm19) deleted mutant HGF052. Meanwhile, two new shunt products, 20-O-demethyl-19-dechloro-N-demethyl-4,5-desepoxy-CAM (4) and 20-O-demethyl-N-demethyl-4,5-desepoxy-CAM (5) were identified from the recombinant strain. Furthermore, by screening of liquid fermentation media, overexpression of bottleneck tailoring enzymes and the pathway-specific activator, the titer of CAM reached 498 mg/L in the engineered strain. Since the 3-O-carbamoyl group of CAM can be removed by chemical cleavage as AP-3 to produce maytansinol, our work suggests that CAM may be a promising alternative to AP-3 in the future development of ADCs.