Antibody-based Cancer Therapy Research Articles

A bispecific antibody that targets the membrane-proximal region of mesothelin and retains high anticancer activity in the presence of shed mesothelin.

Mesothelin (MSLN) is a cell-surface protein that is expressed on many cancers, which makes it a popular target for antibody-based cancer therapy. However, MSLN is shed from cancer cells at high levels via proteases that cleave at its membrane-proximal C-terminal region. Shed MSLN accumulates in patient fluids and tumors and can block antibody-based MSLN-targeting drugs from killing cancer cells. A previously established monoclonal antibody (mAb), 15B6, binds MSLN at its protease-sensitive C-terminal region and does not bind shed MSLN. 15B6 variable fragment (Fv)-derived chimeric antigen receptor (CAR) T cells are not inhibited by shed MSLN and kill tumors in mice more effectively than mAb SS1 Fv-derived CAR T cells, which bind an epitope retained in shed MSLN. Here, we have established 15B6 Fv-derived MSLN x CD3 bispecific antibodies (BsAbs) that target MSLN-expressing cancers. We identified our lead candidate, BsAb 5, after screening multiple 15B6-derived BsAb formats in vitro for cytotoxic activity. BsAb 5 activates T cells to kill various cancer cell lines in a MSLN-specific manner. MSLN 296-591 His, a recombinant protein mimicking shed MSLN, does not inhibit 15B6-derived BsAb 5 but completely inhibits humanized SS1-derived BsAb 7. Furthermore, BsAb 5 inhibits and delays tumor growth and is not inhibited by MSLN 296-585 His in mice. Our findings indicate that by targeting the protease-sensitive region of MSLN, BsAb 5 has high MSLN-specific anticancer activity that is not inhibited by shed MSLN. BsAb 5 may be a promising immunotherapy candidate for MSLN-expressing cancers.

Genetic code expansion in E. coli enables production of a functional ‘ready-to-click’ T cell receptor-specific scFv

Antibody-based cancer therapies have been evolving at a rapid pace in the pharmaceutical market. Bispecific antibody-drug conjugates that engage immune cells to target and kill cancer cells with precision have inspired the development of immunotherapy. Miniaturized antibody fragments such as diabodies, nanobodies, or single-chain variable fragments (scFvs) hold great promise as antibody-drug conjugates as they specifically target tumor tissue and can penetrate it. Here, we optimized the soluble periplasmic expression of the scFv OKT3 comprising the variable VH and VL domains of the mouse anti-human CD3 antibody muromonab-CD3 (trade name Orthoclone OKT3) in E. coli. By an expansion of the genetic code, we site-specifically incorporated the reactive non-canonical amino acid Nε-((2-azidoethoxy)carbonyl)-L-lysine (AzK) into scFv OKT3 using an orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair. To confirm the AzK incorporation and to demonstrate the accessibility of the reactive azide group, we conjugated a fluorophore to scFv OKT3 AzK variants by copper-free strain-promoted alkyne-azide cycloaddition (‘click chemistry’). The scFv OKT3 wild type and the AzK variants bound T cells at nanomolar concentrations. In this study, a ‘ready-to-click’ scFv OKT3 was successfully developed for future applications, e.g. as controlled anti-T cell antibody-drug conjugate or bispecific T cell engager and for imaging immune T cell migration in cancers.

Identification of Membrane-expressed CAPRIN-1 as a Novel and Universal Cancer Target, and Generation of a Therapeutic Anti-CAPRIN-1 Antibody TRK-950.

Antibody-based cancer therapies have been demonstrated to be effective, but are only approved for a limited number of targets, because the majority of these markers is shared with healthy tissue, which may result in adverse effects. Here, we have successfully identified CAPRIN-1 as a novel truly cancer-specific target, universally expressed on membranes of various cancer cells including cancer stem cells. Clinical studies are underway for the anti-CAPRIN-1 therapeutic antibody TRK-950.

Abstract 3300: Development of a diagnostic antibody for anti-BTN1A1 therapy, a novel immune checkpoint inhibitor mutually exclusive of PD-L1/PD-1

Abstract Butyrophilin 1A1 (BTN1A1) is currently undergoing investigation in Phase 1 clinical trials as a novel immune checkpoint protein for the treatment of solid tumors. BTN1A1 exhibits a mutually exclusive expression pattern to that of PD-1/PD-L1, making anti-BTN1A1 antibody therapy an attractive option for cancer patients with PD-L1 refractory or relapsed disease. While current cancer immunotherapies have shown some efficacy against late-stage disease, there remains a major unmet medical need as a large number of patients fail to respond to current treatments. BTN1A1 has been studied at length in preclinical models to characterize its immunomodulatory functions, revealing its ability to inhibit the proliferation of activated T cells and the ability of these cells to kill tumor cells in vitro. In vivo, BTN1A1-overexpressing tumor models exhibit accelerated growth, whereas the growth of BTN1A1-deficient tumors is impaired in immunocompetent settings, supporting a role for this protein in the regulation of immune evasion. Here in, we present the development of a diagnostic antibody for use in the anti-BTN1A1 therapy clinical trials to enable the immunohistochemical (IHC) detection of human BTN1A1. A high-quality BTN1A1 antibody may help predict which patients are more likely to respond to BTN1A1 antibody-based cancer therapy, similar to the PD-L1 companion diagnostic test for anti-PD-L1/PD-1 therapy. The characterization of the developed BTN1A1 antibody was conducted via ELISA, Western blotting, confocal microscopy, and flow cytometry to demonstrate its overall performance. Validation was performed using formalin-fixed paraffin-embedded human tumor tissue samples to determine the analytical sensitivity and specificity of the IHC assay. This antibody was also found to be of value for imaging mass cytometry and flow cytometry experiments elucidating applicable clinical features of both human tumor tissues and immune cells. Specifically, the developed antibody can detect the robust expression of BTN1A1 on both cancer cells and immune cells, while also revealing the mutually exclusive expression of BTN1A1 and PD-L1. Finally, we demonstrate the capability of a BTN1A1 antibody that can be used to identify BTN1A1-positive patients with a high degree of analytical sensitivity and specificity. Citation Format: Stephen S. Yoo. Development of a diagnostic antibody for anti-BTN1A1 therapy, a novel immune checkpoint inhibitor mutually exclusive of PD-L1/PD-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3300.

A cross-reactive pH-dependent EGFR antibody with improved tumor selectivity and penetration obtained by structure-guided engineering

The clinical use of anti-EGFR antibody-based cancer therapy has been limited by antibody-EGFR binding in normal tissues, so developing pH-dependent anti-EGFR antibodies that selectively bind with EGFR in tumors-by taking advantage of the acidity of tumor microenvironment relative to normal tissues-may overcome these limitations. Here, we generated pH-dependent anti-EGFR antibodies with cross-species reactivity for human and mouse EGFR, and we demonstrate that pH-dependent antibodies exhibit tumor-selective binding by binding strongly to EGFR under acidic conditions (pH 6.5) but binding weakly under neutral (pH 7.4) conditions. Based on screening a non-immune human antibody library and antibody affinity maturation, we initially generated antibodies with cross-species reactivity for human and mouse EGFR. A structure model was subsequently constructed and interrogated for hotspots affecting pH-dependent binding, which supported development of a cross-reactive pH-dependent anti-EGFR antibody, G532. Compared with its non-pH-dependent antibody variant, G532 exhibits improved tumor selectivity, tumor penetration, and antitumor activity. Thus, beyond showing that pH-dependentanti-EGFR antibodies can overcome multiple limitationswith antibody-based cancer therapies targeting EGFR, our study illustrates a structure-guided antibody-antigenbinding pH-dependency engineering strategy to enhance antibody tumor selectivity and tumor penetration, which can inform the future development of antibody-based cancer therapies targeting other ubiquitously expressed molecules.

Antibody-Incorporated Nanomedicines for Cancer Therapy.

Antibody-based cancer therapy, one of the most significant therapeutic strategies, has achieved considerable success and progress over the past decades. Nevertheless, obstacles including limited tumor penetration, short circulation half-lives, undesired immunogenicity, and off-target side effects remain to be overcome for the antibody-based cancer treatment. Owing to the rapid development of nanotechnology, antibody-containing nanomedicines that have been extensively explored to overcome these obstacles have already demonstrated enhanced anticancer efficacy and clinical translation potential. This review intends to offer an overview of the advancements of antibody-incorporated nanoparticulate systems in cancer treatment, together with the nontrivial challenges faced by these next-generation nanomedicines. Diverse strategies of antibody immobilization, formats of antibodies, types of cancer-associated antigens, and anticancer mechanisms of antibody-containing nanomedicines are provided and discussed in this review, with an emphasis on the latest applications. The current limitations and future research directions on antibody-containing nanomedicines are also discussed from different perspectives to provide new insights into the construction of anticancer nanomedicines.

An antibody Fc engineered for conditional antibody-dependent cellular cytotoxicity at the low tumor microenvironment pH

Despite the exquisite specificity and high affinity of antibody-based cancer therapies, treatment side effects can occur since the tumor-associated antigens targeted are also present on healthy cells. However, the low pH of the tumor microenvironment provides an opportunity to develop conditionally active antibodies with enhanced tumor specificity. Here, we engineered the human IgG1 Fc domain to enhance pH-selective binding to the receptor FcγRIIIa and subsequent antibody-dependent cellular cytotoxicity (ADCC). We displayed the Fc domain on the surface of mammalian cells and generated a site-directed library by altering Fc residues at the Fc–FcγRIIIa interface to support interactions with positively charged histidine residues. We then used a competitive staining and flow cytometric selection strategy to isolate Fc variants exhibiting reduced FcγRIIIa affinities at neutral pH, but physiological affinities at the tumor-typical pH 6.5. We demonstrate that antibodies composed of Fab arms binding the breast cell epithelial marker Her2 and the lead Fc variant, termed acid-Fc, exhibited an ∼2-fold pH-selectivity for FcγRIIIa binding based on the ratio of equilibrium dissociation constants Kd,7.4/Kd,6.5, due to a faster dissociation rate at pH 7.4. Finally, in vitro ADCC assays with human FcγRIIIa-positive natural killer and Her2-positive target cells demonstrated similar activities for anti-Her2 antibodies bearing the wild-type or acid-Fc at pH 6.5, but nearly 20-fold reduced ADCC for acid-Fc at pH 7.4, based on EC50 ratios. This work shows the promise of mammalian cell display for Fc engineering and the feasibility of pH-selective Fc activation to provide a second dimension of selective tumor cell targeting.

Antibody-based drug delivery systems for cancer therapy: Mechanisms, challenges, and prospects.

In recent years, antibody-based cancer therapy has emerged as one of the efficient therapeutic strategies, such as immune checkpoint inhibitors (ICIs), angiogenesis inhibitors, antibody-drug conjugates (ADCs), multi-specific antibodies, and chimeric antigen receptor T (CAR-T) cells, among others. To date, various drug delivery platforms have been developed to improve the bioavailability, delivery convenience, and reduced toxicity towards increased therapeutic efficacy of antibodies. Herein, we emphasize the clinical manifestations of various antibody-based tumor therapies, highlighting their mechanisms and applications for cancer therapy. Further, based on the problems to be solved in the current clinical application of antibodies, and combined with the advanced drug delivery technologies, we discuss the roles of antibody-based drug delivery systems (DDSs) in cancer therapy, such as enhanced patient compliance and regulating the tumor microenvironment for combined therapy. By expounding the importance of DDSs and discussing the challenges and prospects of their implementation, we suggest that pharmaceutical enterprises and scientists develop appropriate antibody-based delivery platforms.

Transferrin Receptor Serves as a Potential Target for Cancer Therapy

Transferrin receptor (TfR) is a glycoprotein that transfers iron from the extracellular matrix to the intracellular environment. Due to the rapid proliferation need, the demand for iron in cancer cells is much greater than in normal cells, possibly explaining the upregulated TfR expression in cancer cells. The overexpression of TfR and its extracellular accessibility, ability to internalize, and central role in cancer cell make this receptor a potential target for antibody-mediated therapy. The TfR can be targeted indirectly by antibodies conjugated to anti-cancer agents or directly through the use of antibodies that disrupt the function of the receptor and induce Fc-mediated effector functions. This article reviews the developments of antibody-based cancer therapy targeting TfR.

Antibody-based cancer therapy.

Over the past 25 years, antibody therapeutics have emerged as clinically and commercially successful pharmaceuticals, rapidly approaching 100 Food and Drug Administration approvals with combined annual global sales exceeding $100 billion. Nearly half of the marketed antibody therapeutics are used in oncology. These antibody-based cancer therapies can be broken down into three categories based on their different mechanisms of action, i.e. (i) natural properties, (ii) engagement of cytotoxic T cells, and (iii) delivery of cytotoxic payloads. Both natural and engineered properties of the antibody molecule are founded on its highly stable and modular architecture. In this review we provide an overview and outlook of the rapidly evolving landscape of antibody-based cancer therapy.

Periplasmic expression of EGFRvIII extracellular domain as a fusion protein in Escherichia coli NiCo21(DE3)

Epidermal growth factor receptor variant III is a mutant variant of EGFR that has a deletion on its DNA encoding extracellular ligan-binding domain. EGFRvIII is only found in cancer cells and not in normal cells, make it an ideal target as a biomarker for antibody-based cancer therapy. This study performed the expression and characterization of EGFRvIII extracellular domain conjugated with a blue fluorescent protein (BFP) as a fusion protein in Escherichia coli periplasmic space. Endoxylanase signal peptide was employed to guide the recombinant protein through the membrane. IPTG as inducer was added into expression medium with various concentrations of 0; 0.1; 0.25; 0.5; 1 mM, followed by periplasmic extraction using the hypertonic solution. Total proteins and periplasmic proteins were characterized using SDS-PAGE and slot blot analyses. Extracellular domain of EGFRvIII-BFP fusion protein was detected using confocal fluorescence microscopy. This study showed that the ∼43 kDa target protein was successfully expressed on E. coli NiCo21(DE3) periplasmic space with optimum IPTG concentration of 0.1 mM and and showed a blue fluorescence color under the microscope..

Biomaterials-Based Delivery of Therapeutic Antibodies for Cancer Therapy.

Considerable breakthroughs in the treatment of malignant tumors using antibody drugs, especially immunomodulating monoclonal antibodies (mAbs), have been made in the past decade. Despite technological advancements in antibody design and manufacture, multiple challenges face antibody-mediated cancer therapy, such as instability in vivo, poor tumor penetration, limited response rate, and undesirable off-target cytotoxicity. In recent years, an increasing number of biomaterials-based delivery systems have been reported to enhance the antitumor efficacy of antibody drugs. This review summarizes the advances and breakthroughs in integrating biomaterials with therapeutic antibodies for enhanced cancer therapy. A brief introduction to the principal mechanism of antibody-based cancer therapy is first established, and then various antibody immobilization strategies are provided. Finally, the current state-of-the-art in biomaterials-based antibody delivery systems and their applications in cancer treatment are summarized, highlighting how the delivery systems augment the therapeutic efficacy of antibody drugs. The outlook and perspective on biomaterials-based delivery of antitumor antibodies are also discussed.

Ultrasound-Triggered Immunotherapy for Cancer Treatment: An Update.

Over the past few decades, immunotherapy has emerged as a promising therapeutic approach to treat some types of cancer. Moreover, antibody-based cancer therapies can trigger apoptosis and cell growth inhibition to induce immune cell destruction of target cells through antibody-dependent cellular cytotoxicity (ADCC). Nevertheless, immunotherapeutic efficiency is often restricted due to deficient delivery or low accumulation of therapeutic molecules at the tumor site. The development of pegylated liposomes with monoclonal antibodies conjugated to their surfaces (immunoliposomes) and triggered with ultrasound can effectively improve drug accessibility by enhancing cell membrane permeability and drug release. This review summarizes existing traditional cancer treatments and their limitations, emphasizing the recent advancements in ultrasound-triggered immunotherapy.

Natural Killer Cells and Anti-Cancer Therapies: Reciprocal Effects on Immune Function and Therapeutic Response.

Simple SummaryNatural Killer (NK) cells are innate lymphocytes that play an important role in the immune response against cancer. Their activity is controlled by a balance of inhibitory and activating receptors, which in cancer can be skewed to favor their suppression in support of immune escape. It is therefore imperative to find ways to optimize their antitumor functionality. In this review, we explore and discuss how their activity influences, or even mediates, the efficacy of various anti-cancer therapies and, vice versa, how their activity can be affected by these therapies. Knowledge of the mechanisms underlying these observations could provide rationales for combining anti-cancer treatments with strategies enhancing NK cell function in order to improve their therapeutic efficacy.Natural Killer (NK) cells are innate immune cells with the unique ability to recognize and kill virus-infected and cancer cells without prior immune sensitization. Due to their expression of the Fc receptor CD16, effector NK cells can kill tumor cells through antibody-dependent cytotoxicity, making them relevant players in antibody-based cancer therapies. The role of NK cells in other approved and experimental anti-cancer therapies is more elusive. Here, we review the possible role of NK cells in the efficacy of various anti-tumor therapies, including radiotherapy, chemotherapy, and immunotherapy, as well as the impact of these therapies on NK cell function.

Isotype selection for antibody-based cancer therapy.

The clinical application of monoclonal antibodies (mAbs) has revolutionized the field of cancer therapy, as it has enabled the successful treatment of previously untreatable types of cancer. Different mechanisms play a role in the anti-tumour effect of mAbs. These include blocking of tumour-specific growth factor receptors or of immune modulatory molecules as well as complement and cell-mediated tumour cell lysis. Thus, for many mAbs, Fc-mediated effector functions critically contribute to the efficacy of treatment. As immunoglobulin (Ig) isotypes differ in their ability to bind to Fc receptors on immune cells as well as in their ability to activate complement, they differ in the immune responses they activate. Therefore, the choice of antibody isotype for therapeutic mAbs is dictated by its intended mechanism of action. Considering that clinical efficacy of many mAbs is currently achieved only in subsets of patients, optimal isotype selection and Fc optimization during antibody development may represent an important step towards improved patient outcome. Here, we discuss the current knowledge of the therapeutic effector functions of different isotypes and Fc-engineering strategies to improve mAbs application.

Monoclonal antibody-based cancer therapies

Targeted therapy has been widely demonstrated as an effective strategy to treat cancers, the leading cause of death in the world. This minireview summarizes the technical platforms and methodologies utilized to develop and engineer therapeutic monoclonal antibodies and antibody-drug conjugates. First, the USA FDA approved monoclonal antibody (mAb)-based targeted therapies are reviewed. Then the representative innovative chimeric, humanized and fully human anti-cancer antibodies and antibody-drug conjugates are described. Finally, the past and predictive market trend of therapeutic antibodies is discussed.

Amivantamab (JNJ-61186372), an Fc Enhanced EGFR/cMet Bispecific Antibody, Induces Receptor Downmodulation and Antitumor Activity by Monocyte/Macrophage Trogocytosis.

Small molecule inhibitors targeting mutant EGFR are standard of care in non-small cell lung cancer (NSCLC), but acquired resistance invariably develops through mutations in EGFR or through activation of compensatory pathways such as cMet. Amivantamab (JNJ-61186372) is an anti-EGFR and anti-cMet bispecific low fucose antibody with enhanced Fc function designed to treat tumors driven by activated EGFR and/or cMet signaling. Potent in vivo antitumor efficacy is observed upon amivantamab treatment of human tumor xenograft models driven by mutant activated EGFR, and this activity is associated with receptor downregulation. Despite these robust antitumor responses in vivo, limited antiproliferative effects and EGFR/cMet receptor downregulation by amivantamab were observed in vitro Interestingly, in vitro addition of isolated human immune cells notably enhanced amivantamab-mediated EGFR and cMet downregulation, leading to antibody dose-dependent cancer cell killing. Through a comprehensive assessment of the Fc-mediated effector functions, we demonstrate that monocytes and/or macrophages, through trogocytosis, are necessary and sufficient for Fc interaction-mediated EGFR/cMet downmodulation and are required for in vivo antitumor efficacy. Collectively, our findings represent a novel Fc-dependent macrophage-mediated antitumor mechanism of amivantamab and highlight trogocytosis as an important mechanism of action to exploit in designing new antibody-based cancer therapies.

Abstract 4436: Combination therapy with an antibody specific for transferrin receptor 1 (ch128.1/IgG1) and bortezomib or lenalidomide results in increased survival in an in vivo model of human multiple myeloma

Abstract Multiple myeloma (MM) is a B-cell proliferative disorder of monoclonal malignant plasma cells. It is the second most common hematologic malignancy in the United States, after non-Hodgkin lymphoma (NHL). Clinical manifestations of MM include osteolytic bone lesions, hypercalcemia, renal failure, anemia, recurrent infections, and neuropathy. Standard treatments for MM include the proteasome inhibitor bortezomib, as well as lenalidomide, a thalidomide analog with immunomodulatory and antiangiogenic properties. Although the use of these two agents in combination with other therapies have significantly improved MM survival, the disease remains incurable. We previously developed a mouse/human chimeric antibody (ch128.1/IgG1) specific for human transferrin receptor 1 (TfR1). TfR1, also known as CD71, is a meaningful target for antibody-based cancer therapy, given its high expression on malignant cells and relevant role in cancer cell pathology. We have reported that treatment of SCID-Beige mice bearing disseminated human KMS-11, MM.1S, or MM.1R myeloma tumors with ch128.1/IgG1 results in significant antitumor activity in early and late disease stages. The mechanism of antitumor activity is Fc-mediated in which macrophages are considered to play a relevant role. Since combining multiple treatments with differing mechanisms of action is an effective antitumor strategy, and given the relevance of bortezomib and lenalidomide in MM therapy, we decided to explore, for the first time, the combination of these two agents with ch128.1/IgG1. We found that treatment with a single dose of ch128.1/IgG1, or multiple doses of bortezomib or lenalidomide used as single agents, results in significant antitumor activity in SCID-Beige mice bearing late stage disseminated human MM.1S tumors. However, this antitumor activity is superior when ch128.1/IgG1 was combined with bortezomib or lenalidomide, showing significantly longer survival compared to each therapy used alone. These results suggest that the addition of a ch128.1/IgG1 therapy arm in a combinatorial treatment regimen involving bortezomib and/or lenalidomide is a promising strategy against human B-cell malignancies such as MM. Citation Format: Pierre V. Candelaria, Miguel Nava, Otoniel Martinez-Maza, Tracy R. Daniels-Wells, Manuel L. Penichet. Combination therapy with an antibody specific for transferrin receptor 1 (ch128.1/IgG1) and bortezomib or lenalidomide results in increased survival in an in vivo model of human multiple myeloma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4436.

Current Perspectives on 89Zr-PET Imaging.

89Zr is an emerging radionuclide that plays an essential role in immuno-positron emission tomography (PET) imaging. The long half-life of 89Zr (t1/2 = 3.3 days) is favorable for evaluating the in vivo distribution of monoclonal antibodies. Thus, the use of 89Zr is promising for monitoring antibody-based cancer therapies. Immuno-PET combines the sensitivity of PET with the specificity of antibodies. A number of studies have been conducted to investigate the feasibility of 89Zr immuno-PET imaging for predicting the efficacy of radioimmunotherapy and antibody therapies, imaging target expression, detecting target-expressing tumors, and the monitoring of anti-cancer chemotherapies. In this review, we summarize the current status of PET imaging using 89Zr in both preclinical and clinical studies by highlighting the use of immuno-PET for the targets of high clinical relevance. We also present 89Zr-PET applications other than immuno-PET, such as nanoparticle imaging and cell tracking. Finally, we discuss the limitations and the ongoing research being performed to overcome the remaining hurdles.

Affinity maturation, humanization, and co-crystallization of a rabbit anti-human ROR2 monoclonal antibody for therapeutic applications

Antibodies are widely used as cancer therapeutics, but their current use is limited by the low number of antigens restricted to cancer cells. A receptor tyrosine kinase, receptor tyrosine kinase-like orphan receptor 2 (ROR2), is normally expressed only during embryogenesis and is tightly down-regulated in postnatal healthy tissues. However, it is up-regulated in a diverse set of hematologic and solid malignancies, thus ROR2 represents a candidate antigen for antibody-based cancer therapy. Here we describe the affinity maturation and humanization of a rabbit mAb that binds human and mouse ROR2 but not human ROR1 or other human cell-surface antigens. Co-crystallization of the parental rabbit mAb in complex with the human ROR2 kringle domain (hROR2-Kr) guided affinity maturation by heavy-chain complementarity-determining region 3 (HCDR3)-focused mutagenesis and selection. The affinity-matured rabbit mAb was then humanized by complementarity-determining region (CDR) grafting and framework fine tuning and again co-crystallized with hROR2-Kr. We show that the affinity-matured and humanized mAb retains strong affinity and specificity to ROR2 and, following conversion to a T cell-engaging bispecific antibody, has potent cytotoxicity toward ROR2-expressing cells. We anticipate that this humanized affinity-matured mAb will find application for antibody-based cancer therapy of ROR2-expressing neoplasms.