Effector-attenuating Substitutions That Maintain Antibody Stability and Reduce Toxicity in Mice.

The antibody Fc region regulates antibody cytotoxic activities and serum half-life. In a therapeutic context, however, the cytotoxic effector function of an antibody is often not desirable and can create safety liabilities by activating native host immune defenses against cells expressing the receptor antigens. Several amino acid changes in the Fc region have been reported to silence or reduce the effector function of antibodies. These earlier studies focused primarily on the interaction of human antibodies with human Fc-γ receptors, and it remains largely unknown how such changes to Fc might translate to the context of a murine antibody. We demonstrate that the commonly used N297G (NG) and D265A, N297G (DANG) variants that are efficacious in attenuating effector function in primates retain potent complement activation capacity in mice, leading to safety liabilities in murine studies. In contrast, we found an L234A, L235A, P329G (LALA-PG) variant that eliminates complement binding and fixation as well as Fc-γ-dependent, antibody-dependent, cell-mediated cytotoxity in both murine IgG2a and human IgG1. These LALA-PG substitutions allow a more accurate translation of results generated with an "effectorless" antibody between mice and primates. Further, we show that both human and murine antibodies containing the LALA-PG variant have typical pharmacokinetics in rodents and retain thermostability, enabling efficient knobs-into-holes bispecific antibody production and a robust path to generating highly effector-attenuated bispecific antibodies for preclinical studies.

A neutralizing anti-gH/gL monoclonal antibody is protective in the guinea pig model of congenital CMV infection.

Human cytomegalovirus (HCMV) is the most common cause of congenital virus infection. Congenital HCMV infection occurs in 0.2-1% of all births, and causes birth defects and developmental abnormalities, including sensorineural hearing loss and developmental delay. Several key studies have established the guinea pig as a tractable model for the study of congenital HCMV infection and have shown that polyclonal antibodies can be protective. In this study, we demonstrate that an anti-guinea pig CMV (GPCMV) glycoprotein H/glycoprotein L neutralizing monoclonal antibody protects against fetal infection and loss in the guinea pig. Furthermore, we have delineated the kinetics of GPCMV congenital infection, from maternal infection (salivary glands, seroconversion, placenta) to fetal infection (fetus and amniotic fluid). Our studies support the hypothesis that a neutralizing monoclonal antibody targeting an envelope GPCMV glycoprotein can protect the fetus from infection and may shed light on the therapeutic intervention of HCMV congenital infection in humans.

Oncogenic RAS pathway activation promotes resistance to anti-VEGF therapy through G-CSF-induced neutrophil recruitment.

Granulocyte-colony stimulating factor (G-CSF) promotes mobilization of CD11b(+)Gr1(+) myeloid cells and has been implicated in resistance to anti-VEGF therapy in mouse models. High G-CSF production has been associated with a poor prognosis in cancer patients. Here we show that activation of the RAS/MEK/ERK pathway regulates G-CSF expression through the Ets transcription factor. Several growth factors induced G-CSF expression by a MEK-dependent mechanism. Inhibition of G-CSF release with a MEK inhibitor markedly reduced G-CSF production in vitro and synergized with anti-VEGF antibodies to reduce CD11b(+)Ly6G(+) neutrophil mobilization and tumor growth and led to increased survival in animal models of cancer, including a genetically engineered mouse model of pancreatic adenocarcinoma. Analysis of biopsies from pancreatic cancer patients revealed increased phospho-MEK, G-CSF, and Ets expression and enhanced neutrophil recruitment compared with normal pancreata. These results provide insights into G-CSF regulation and on the mechanism of action of MEK inhibitors and point to unique anticancer strategies.

Real-time immuno-polymerase chain reaction in a 384-well format: detection of vascular endothelial growth factor and epidermal growth factor-like domain 7.

Immuno-polymerase chain reaction (immuno-PCR) combines the specificity of antibodies with the amplification power of PCR to detect low levels of proteins. Here, we describe the development of a 384-well immuno-PCR method that uses streptavidin coated on a PCR plate to capture complexes of biotinylated capture antibody, antigen, and DNA-labeled detection antibody. Unbound molecules are removed by a wash step using a standard plate washer. Antibody-DNA molecules in bound complexes are then detected directly on the plate using real-time PCR. Circulating human vascular endothelial growth factor concentrations measured by this method correlated with measurements obtained from enzyme-linked immunosorbent assay (ELISA). Using this method, we developed an assay for human epidermal growth factor-like domain 7 (EGFL7), an extracellular matrix-bound angiogenic factor. EGFL7 is expressed at a higher level in certain cancers, although endogenous EGFL7 concentrations have not been reported. Our 384-well EGFL7 immuno-PCR assay can detect 0.51pM EGFL7 in plasma, approximately 16-fold more sensitive than the ELISA, utilizing the same antibodies. This assay detected EGFL7 in lysates of non-small-cell lung cancer and hepatocellular carcinoma cell lines and also hepatocellular carcinoma, breast cancer, and ovarian cancer tissues. This 384-well immuno-PCR method can be used to develop high-throughput biomarker assays.

Bv8 regulates myeloid-cell-dependent tumour angiogenesis.

Bone-marrow-derived cells facilitate tumour angiogenesis, but the molecular mechanisms of this facilitation are incompletely understood. We have previously shown that the related EG-VEGF and Bv8 proteins, also known as prokineticin 1 (Prok1) and prokineticin 2 (Prok2), promote both tissue-specific angiogenesis and haematopoietic cell mobilization. Unlike EG-VEGF, Bv8 is expressed in the bone marrow. Here we show that implantation of tumour cells in mice resulted in upregulation of Bv8 in CD11b+Gr1+ myeloid cells. We identified granulocyte colony-stimulating factor as a major positive regulator of Bv8 expression. Anti-Bv8 antibodies reduced CD11b+Gr1+ cell mobilization elicited by granulocyte colony-stimulating factor. Adenoviral delivery of Bv8 into tumours was shown to promote angiogenesis. Anti-Bv8 antibodies inhibited growth of several tumours in mice and suppressed angiogenesis. Anti-Bv8 treatment also reduced CD11b+Gr1+ cells, both in peripheral blood and in tumours. The effects of anti-Bv8 antibodies were additive to those of anti-Vegf antibodies or cytotoxic chemotherapy. Thus, Bv8 modulates mobilization of CD11b+Gr1+ cells from the bone marrow during tumour development and also promotes angiogenesis locally.

Granulocyte-colony stimulating factor promotes lung metastasis through mobilization of Ly6G+Ly6C+ granulocytes.

Priming of the organ-specific premetastatic sites is thought to be an important yet incompletely understood step during metastasis. In this study, we show that the metastatic tumors we examined overexpress granulocyte-colony stimulating factor (G-CSF), which expands and mobilizes Ly6G+Ly6C+ granulocytes and facilitates their subsequent homing at distant organs even before the arrival of tumor cells. Moreover, G-CSF-mobilized Ly6G+Ly6C+ cells produce the Bv8 protein, which has been implicated in angiogenesis and mobilization of myeloid cells. Anti-G-CSF or anti-Bv8 antibodies significantly reduced lung metastasis. Transplantation of Bv8 null fetal liver cells into lethally irradiated hosts also reduced metastasis. We identified an unexpected role for Bv8: the ability to stimulate tumor cell migration through activation of one of the Bv8 receptors, prokineticin receptor (PKR)-1. Finally, we show that administration of recombinant G-CSF is sufficient to increase the numbers of Ly6G+Ly6C+ cells in organ-specific metastatic sites and results in enhanced metastatic ability of several tumors.

Real-time quantification of antibody-short interfering RNA conjugate in serum by antigen capture reverse transcription-polymerase chain reaction.

Short interfering RNA (siRNA) has therapeutic potential. However, efficient delivery is a formidable task. To facilitate delivery of siRNA into cells, we covalently conjugated siRNA to antibodies that bind to cell surface proteins and internalize. Understanding how these antibody-siRNA conjugates function in vivo requires pharmacokinetic analysis. Thus, we developed a simple real-time antigen capture reverse transcription-polymerase chain reaction (RT-PCR) assay to detect intact antibody-siRNA conjugates. Biotinylated antigen bound to streptavidin-coated PCR tubes was used to capture antibody-siRNA conjugate. The captured antibody-siRNA conjugate was then reverse-transcribed in the same tube, avoiding a sample transfer step. This reproducible assay had a wide standard curve range of 0.029 to 480ng/ml and could detect as low as 0.58ng/ml antibody-siRNA conjugates in mouse serum. The presence of unconjugated antibody that could be generated from siRNA degradation in vivo did not affect the assay as long as the total antibody concentration in the antigen capture step did not exceed 480ng/ml. Using this assay, we observed a more rapid decrease in serum antibody-siRNA conjugate concentrations than the total antibody concentrations in mice dosed with antibody-siRNA conjugates, suggesting loss of siRNA from the antibody. This assay is useful for optimizing antibody-siRNA and likely aptamer-siRNA conjugates to improve pharmacokinetics and aid siRNA delivery.

G-CSF-initiated myeloid cell mobilization and angiogenesis mediate tumor refractoriness to anti-VEGF therapy in mouse models.

Recent studies suggest that tumor-associated CD11b(+)Gr1(+) myeloid cells contribute to refractoriness to antiangiogenic therapy with an anti-VEGF-A antibody. However, the mechanisms of peripheral mobilization and tumor-homing of CD11b(+)Gr1(+) cells are unclear. Here, we show that, compared with other cytokines [granulocyte-macrophage colony stimulating factor (GM-CSF), stromal derived factor 1alpha, and placenta growth factor], G-CSF and the G-CSF-induced Bv8 protein have preferential expression in refractory tumors. Treatment of refractory tumors with the combination of anti-VEGF and anti-G-CSF (or anti-Bv8) reduced tumor growth compared with anti-VEGF-A monotherapy. Anti-G-CSF treatment dramatically suppressed circulating or tumor-associated CD11b(+)Gr1(+) cells, reduced Bv8 levels, and affected the tumor vasculature. Conversely, G-CSF delivery to animals bearing anti-VEGF sensitive tumors resulted in reduced responsiveness to anti-VEGF-A treatment through induction of Bv8-dependent angiogenesis. We conclude that, at least in the models examined, G-CSF expression by tumor or stromal cells is a determinant of refractoriness to anti-VEGF-A treatment.

Direct Tie2 Agonists Stabilize Vasculature for the Treatment of Diabetic Macular Edema.

Purpose: Diabetic macular edema (DME) is the leading cause of vision loss and blindness among working-age adults. Although current intravitreal anti-vascular endothelial growth factor (VEGF) therapies improve vision for many patients with DME, approximately half do not achieve the visual acuity required to drive. We therefore sought additional approaches to resolve edema and improve vision for these patients. Methods: We explored direct agonists of Tie2, a receptor known to stabilize vasculature and prevent leakage. We identified a multivalent PEG-Fab conjugate, Tie2.1-hexamer, that oligomerizes Tie2 and drives receptor activation and characterized its activities in vitro and in vivo. Results: Tie2.1-hexamer normalized and stabilized intercellular junctions of stressed endothelial cell monolayers in vitro, suppressed vascular leak in mice under conditions where anti-VEGF alone was ineffective, and demonstrated extended ocular exposure and robust pharmacodynamic responses in non-human primates. Conclusions: Tie2.1-hexamer directly activates the Tie2 pathway, reduces vascular leak, and is persistent within the vitreal humor. Translational Relevance: Our study presents a promising potential therapeutic for the treatment of DME.

TGFß2 and TGFß3 isoforms drive fibrotic disease pathogenesis.

Transforming growth factor-ß (TGFß) is a key driver of fibrogenesis. Three TGFß isoforms (TGFß1, TGFß2, and TGFß3) in mammals have distinct functions in embryonic development; however, the postnatal pathological roles and activation mechanisms of TGFß2 and TGFß3 have not been well characterized. Here, we show that the latent forms of TGFß2 and TGFß3 can be activated by integrin-independent mechanisms and have lower activation thresholds compared to TGFß1. Unlike TGFB1, TGFB2 and TGFB3 expression is increased in human lung and liver fibrotic tissues compared to healthy control tissues. Thus, TGFß2 and TGFß3 may play a pathological role in fibrosis. Inducible conditional knockout mice and anti-TGFß isoform-selective antibodies demonstrated that TGFß2 and TGFß3 are independently involved in mouse fibrosis models in vivo, and selective TGFß2 and TGFß3 inhibition does not lead to the increased inflammation observed with pan-TGFß isoform inhibition. A cocrystal structure of a TGFß2-anti-TGFß2/3 antibody complex reveals an allosteric isoform-selective inhibitory mechanism. Therefore, inhibiting TGFß2 and/or TGFß3 while sparing TGFß1 may alleviate fibrosis without toxicity concerns associated with pan-TGFß blockade.

In vivo blockade of OX40 ligand inhibits thymic stromal lymphopoietin driven atopic inflammation.

Thymic stromal lymphopoietin (TSLP) potently induces deregulation of Th2 responses, a hallmark feature of allergic inflammatory diseases such as asthma, atopic dermatitis, and allergic rhinitis. However, direct downstream in vivo mediators in the TSLP-induced atopic immune cascade have not been identified. In our current study, we have shown that OX40 ligand (OX40L) is a critical in vivo mediator of TSLP-mediated Th2 responses. Treating mice with OX40L-blocking antibodies substantially inhibited immune responses induced by TSLP in the lung and skin, including Th2 inflammatory cell infiltration, cytokine secretion, and IgE production. OX40L-blocking antibodies also inhibited antigen-driven Th2 inflammation in mouse and nonhuman primate models of asthma. This treatment resulted in both blockade of the OX40-OX40L receptor-ligand interaction and depletion of OX40L-positive cells. The use of a blocking, OX40L-specific mAb thus presents a promising strategy for the treatment of allergic diseases associated with pathologic Th2 immune responses.

Characterization and regulation of bv8 in human blood cells.

PURPOSE: Bv8, also known as prokineticin 2, has been recently shown to be a mediator of myeloid cell-dependent tumor angiogenesis in mouse models. We wished to determine whether these findings might be potentially relevant to human disease. EXPERIMENTAL DESIGN: We characterized Bv8 expression in human blood cells in vitro and in vivo, and did Bv8 immunohistochemistry in human tumor sections. We also partially purified Bv8 from human neutrophils and tested its bioactivity. RESULTS: We found that Bv8 expression is regulated by several cytokines in a cell type-specific fashion. Both granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor induced Bv8 expression in neutrophils and bone marrow cells, whereas interleukin 10 up-regulated Bv8 expression in monocytes and lymphocytes. Bv8 potently promoted neutrophil chemotaxis. Bv8 protein isolated from human neutrophils was found to be biologically active. Of the two receptors for Bv8 [prokineticin receptor 1(PKR1)/endocrine gland-derived vascular endothelial growth factor receptor 1 (EG-VEGFR1) and PKR2/EG-VEGFR2], only PKR2/EG-VEGFR2 was detectable in human neutrophils. Also, we found a marked up-regulation of Bv8 mRNA and protein in peripheral blood mononuclear cells from G-CSF-treated donors compared with those from untreated individuals, verifying our in vitro observations. Finally, immunohistochemistry showed Bv8 expression in neutrophils infiltrating human tumors. CONCLUSIONS: These results provide the basis for further investigation of the pathophysiologic role of Bv8 in human tumors and inflammatory disorders and, potentially, for therapeutic application of Bv8 inhibitors.

Reproducible quantification of IgG uptake at endogenous and overexpressed FcRn levels at pH 7.4: Comparison of a wild type IgG and a stronger FcRn binding variant.

IgG antibodies have been used to treat many diseases including cancer. IgG antibody-drug conjugates (ADCs) deliver cytotoxic drugs to target cells for cell elimination, but they have dose limiting toxicity due to target-independent uptake, including pinocytotic uptake. Neonatal Fc receptor (FcRn) recycles pinocytosed IgG in a pH-dependent manner and is the receptor responsible for the long half-life of IgG. Use of IgG variants with stronger FcRn binding at pH 6.0 for ADCs might improve recycling efficiency and reduce toxicity. However, these variants have residual FcRn binding at pH 7.4, which could lead to FcRn-mediated uptake and higher toxicity. Thus, the uptake of such variants at pH 7.4 needs to be evaluated. Here we report a reproducible and quantitative assay using an inducible HM7 colorectal cancer cell line to measure IgG uptake at endogenous and overexpressed FcRn levels. Our assay had comparable reproducibility at pH 6.0, 6.8 and 7.4. The wild type (WT) IgG had similar uptake at endogenous and overexpressed FcRn levels, as expected for pinocytotic uptake. We found similar uptake of a WT IgG and a stronger FcRn binding T307Q/N434A variant (QA variant) at endogenous FcRn levels at pH 7.4, although the QA variant had higher uptake at overexpressed FcRn levels. The QA variant also had higher uptake than the WT IgG at overexpressed FcRn levels at pH 6.8. Our assay can be used to characterize the stronger FcRn binding variants to aid in selection of suitable variants with low uptake at pH 7.4 for use as ADCs.

Complex formation of anti-VEGF-C with VEGF-C released during blood coagulation resulted in an artifact in its serum pharmacokinetics.

A phage-derived human monoclonal antibody against VEGF-C was developed as a potential anti-tumor therapeutic and exhibited fast clearance in preclinical species, with notably faster clearance in serum than in plasma. The purpose of this work was to understand the factors contributing to its fast clearance. In vitro incubations in animal and human blood, plasma, and serum were conducted with radiolabeled anti-VEGF-C to determine potential protein and cell-based interactions with the antibody as well as any matrix-dependent recovery dependent upon the matrix. A tissue distribution study was conducted in mice with and without heparin infusion in order to identify a tissue sink and determine whether heparin could affect antibody recovery from serum and/or plasma. Incubation of radiolabeled anti-VEGF-C in human and animal blood, plasma, or serum revealed that the antibody formed a complex with an endogenous protein, likely VEGF-C. This complex was trapped within the blood clot during serum preparation from blood, but not within the blood cell pellet during plasma preparation. Low level heparin infusion in mice slowed down clot formation during serum preparation and allowed for better recovery of the radiolabeled antibody in serum. No tissue sink was found in mice. Thus, during this characterization, we determined that the blood sampling matrix greatly impacted the amount of antibody recovered in the samples, therefore, altering its derived pharmacokinetic parameters. Target biology should be considered when selecting appropriate sampling matrices for PK analysis.

A Potent Pan-TGFß Neutralizing Monoclonal Antibody Elicits Cardiovascular Toxicity in Mice and Cynomolgus Monkeys.

Transforming growth factor ß (TGFß) signaling has been recently shown to reduce antitumor response to PD-L1 blockade, leading to a renewed enthusiasm in developing anti-TGFß therapies for potential combination with cancer immunotherapy agents. Inhibition of TGFß signaling in nonclinical toxicology species is associated with serious adverse toxicities including cardiac valvulopathies and anemia. Previously, cardiovascular toxicities have been thought to be limited to small molecule inhibitors of TGFß receptor and not considered to be a liability associated with pan-TGFß neutralizing monoclonal antibodies (mAbs). Here, we report the toxicity findings associated with a potent pan-TGFß neutralizing mAb (pan-TGFß mAb; neutralizes TGFß1, 2, and 3) after 5 weekly intravenous doses of 10, 30, and 100 mg/kg, followed by a 4-week recovery period, in mice and cynomolgus monkeys. Mortality was observed due to acute bleeding and cardiovascular toxicity in mice at ≥ 30 mg/kg and prolonged menstruation in female monkeys at 100 mg/kg. Additional findings considered to be on-target exaggerated pharmacology included generalized bleeding and cardiovascular toxicity in mice and monkeys; histopathologic changes in the teeth, tongue, and skin in mice; and abnormal wound healing and microscopic pathology in the bone in monkeys. Importantly, our data indicate that the cardiovascular toxicities associated with the inhibition of TGFß signaling are not limited to small molecule inhibitors but are also observed following administration of a potent pan-TGFß inhibiting mAb.

Massive antibody discovery used to probe structure-function relationships of the essential outer membrane protein LptD.

Outer membrane proteins (OMPs) in Gram-negative bacteria dictate permeability of metabolites, antibiotics, and toxins. Elucidating the structure-function relationships governing OMPs within native membrane environments remains challenging. We constructed a diverse library of >3000 monoclonal antibodies to assess the roles of extracellular loops (ECLs) in LptD, an essential OMP that inserts lipopolysaccharide into the outer membrane of Escherichia coli. Epitope binning and mapping experiments with LptD-loop-deletion mutants demonstrated that 7 of the 13 ECLs are targeted by antibodies. Only ECLs inaccessible to antibodies were required for the structure or function of LptD. Our results suggest that antibody-accessible loops evolved to protect key extracellular regions of LptD, but are themselves dispensable. Supporting this hypothesis, no α-LptD antibody interfered with essential functions of LptD. Our experimental workflow enables structure-function studies of OMPs in native cellular environments, provides unexpected insight into LptD, and presents a method to assess the therapeutic potential of antibody targeting.

Electrochemiluminescence to detect surface proteins on live cells.

Flow cytometry and cell-based enzyme-linked immunosorbent assays have been used to detect surface proteins on cells. A recently available electrochemiluminescent assay methodology using carbon surface electrodes built into the bottom of microwell plates can be used as an alternative to these methods and has some advantages. The carbon surface plates bind suspension cells tightly enough to allow plates to be washed using a plate washer. This eliminates the centrifugation steps typically used for washing suspension cells. For adherent cells, human umbilical vein endothelial cells can be grown, activated, and assayed in the same carbon surface plate. This eliminates the need to detach cells from tissue culture plates for analysis, making the electrochemiluminescent assay much easier to perform than a corresponding flow cytometry assay. This electrochemiluminescence technology provides a high throughput method to detect surface proteins on live cells.

A targeted boost-and-sort immunization strategy using Escherichia coli BamA identifies rare growth inhibitory antibodies.

Outer membrane proteins (OMPs) in Gram-negative bacteria are essential for a number of cellular functions including nutrient transport and drug efflux. Escherichia coli BamA is an essential component of the OMP ß-barrel assembly machinery and a potential novel antibacterial target that has been proposed to undergo large (~15 Å) conformational changes. Here, we explored methods to isolate anti-BamA monoclonal antibodies (mAbs) that might alter the function of this OMP and ultimately lead to bacterial growth inhibition. We first optimized traditional immunization approaches but failed to identify mAbs that altered cell growth after screening >3000 hybridomas. We then developed a "targeted boost-and-sort" strategy that combines bacterial cell immunizations, purified BamA protein boosts, and single hybridoma cell sorting using amphipol-reconstituted BamA antigen. This unique workflow improves the discovery efficiency of FACS + mAbs by >600-fold and enabled the identification of rare anti-BamA mAbs with bacterial growth inhibitory activity in the presence of a truncated lipopolysaccharide layer. These mAbs represent novel tools for dissecting the BamA-mediated mechanism of ß-barrel folding and our workflow establishes a new template for the efficient discovery of novel mAbs against other highly dynamic membrane proteins.

A high throughput electrochemiluminescent cell-binding assay for therapeutic anti-CD20 antibody selection.

A cell-based ELISA using suspension WIL2 cells in 96-well format was previously developed for measuring relative binding affinities of humanized anti-CD20 variants. We further developed a new cell-binding assay that uses high binding capacity carbon electrode plates for rapid attachment of suspension WIL2 cells and electrochemiluminescence for detection. Compared to the cell-based ELISA, which requires centrifugation for the manual wash steps, significant improvement in assay throughput was achieved by using a microplate washer. The assay can be performed on both 96- and 384-well plates with a standard curve range of 2.74-2000 ng/ml, which is wider than the range of 15.6-1000 ng/ml for the cell-based ELISA. Using CD20 expressing CHO cell clones, surface expression of >or=33,000 CD20 molecules was sufficient to obtain a dose-response curve in 384-well format. Relative affinities of 15 humanized variants correlated well (r(2)=0.94) between electrochemiluminescent cell-binding assay and cell-based ELISA. A competitive assay format, using mouse anti-CD20 antibody as the tracer, with a dose-response range of 27.4-20,000 ng/ml was also developed. The new cell-binding assay method can be used to efficiently support humanization process for selection of anti-CD20 antibody drug candidates and to characterize antibody binding to other cell surface proteins.

Evading pre-existing anti-hinge antibody binding by hinge engineering.

Antigen-binding fragments (Fab) and F(ab')2 antibodies serve as alternative formats to full-length anti-bodies in therapeutic and immune assays. They provide the advantage of small size, short serum half-life, and lack of effector function. Several proteases associated with invasive diseases are known to cleave antibodies in the hinge-region, and this results in anti-hinge antibodies (AHA) toward the neoepitopes. The AHA can act as surrogate Fc and reintroduce the properties of the Fc that are otherwise lacking in antibody fragments. While this response is desired during the natural process of fighting disease, it is commonly unwanted for therapeutic antibody fragments. In our study, we identify a truncation in the lower hinge region of the antibody that maintains efficient proteolytic cleavage by IdeS protease. The resulting neoepitope at the F(ab')2 C-terminus does not have detectable binding of pre-existing AHA, providing a practical route to produce F(ab')2 in vitro by proteolytic digestion when the binding of pre-existing AHA is undesired. We extend our studies to the upper hinge region of the antibody and provide a detailed analysis of the contribution of C-terminal residues of the upper hinge of human IgG1, IgG2 and IgG4 to pre-existing AHA reactivity in human serum. While no pre-existing antibodies are observed toward the Fab of IgG2 and IgG4 isotype, a significant response is observed toward most residues of the upper hinge of human IgG1. We identify a T225L variant and the natural C-terminal D221 as solutions with minimal serum reactivity. Our work now enables the production of Fab and F(ab')2 for therapeutic and diagnostic immune assays that have minimal reactivity toward pre-existing AHA.