Preclinical Studies into Specific Toxicity of a Candidate Drug Based on Complement C3 neodeterminant Recombinant Humanized Antibody for Treating Traumatic Brain Injuries

This study aims to assess the specific toxicity (allergenicity, immunotoxicity and reproduction toxicity) of a dosage form of complement C3 neodeterminant recombinant humanized antibody. The reactions of general anaphylaxis, active cutaneous anaphylaxis and delayed-type reaction demonstrated no drug-related signs of experimental animal sensitization and immediate or delayed hypersensitivity in the treated animals. It was found that, in doses exceeding significantly the expected human therapeutic dose, the drug has no effect on humoral and cell-mediated immune responses. In addition, the drug does not suppress the phagocytic cell activity thereby indicating the absence of immunotoxic effect. Moreover, the drug has no adverse effects on male and female reproduction, progeny embryonal development, as well as prenatal and postnatal progeny development. The obtained data can be used in future clinical studies of the drug safety.

891 S-531011, a novel anti-human CCR8 antibody: antibody screening and evaluation of biological profiles

BackgroundRegulatory T cells (Tregs) are involved in tumor progression and inhibition of anti-tumor immune responses by promotion of immunological tolerance in the tumor microenvironment. On the other hand, Treg cells in peripheral blood are also essential role in preventing autoimmunity and uncontrolled inflammation. So, selective control of tumor infiltrating Treg cells might be an attractive approach of immune-oncology therapies without disrupting their systemic anti-inflammatory functions. Here, we focused on CCR8 (C-C motif chemokine receptor 8) as a target molecule which was selectively and highly expressed on tumor-infiltrating Tregs and developed a novel anti-human CCR8 specific antibody.MethodsWe immunized mice with human CCR8 by our original immunization method which could strongly induce antibodies for membrane proteins, and then constructed hybridoma cells. Anti-human CCR8 (human CCR4 as a negative control) binding assay and human CCL1-CCR8 neutralizing assay were simultaneously performed by using supernatants of hybridoma cells to isolate human CCR8 specific strong-neutralizing antibodies. After humanization and affinity maturation of some selected clones, we selected our lead antibody by binding specificity, neutralizing activity, antibody dependent cellular cytotoxicity (ADCC) and thermodynamic stability as index.ResultsWe rapidly induced human CCR8 specific antibodies in mouse with our unique immunization methods and constructed thousands of hybridomas secreting anti-human CCR8 antibodies. We also successfully humanized some of lead antibodies which show high affinity and specificity and isolated novel anti-human CCR8 specific humanized antibody S-531011 as our development antibody after affinity maturation. S-531011 selectively recognizes human CCR8 on the surface of tumor-infiltrating Tregs and shows strong ADCC. While human CCL1 is known as a dominant ligand of CCR8 which binds extracellular loop2 and N-terminal of CCR8, S-531011 recognizes similar epitopes and effectively neutralizes CCL1-CCR8 signaling. Furthermore, S-531011 also shows favorable blood kinetics in vivo and potently inhibits tumor growth in tumor bearing human CCR8 knock-in mouse model.ConclusionsWe develop S-531011, a novel anti-human CCR8 humanized antibody which could selectively recognize and deplete tumor infiltrating Tregs. Based on our pre-clinical data, S-531011 has strong anti-tumor effect and we expect that it might be a potent novel tumor immuno-therapeutic agent with fewer side effect.Ethics ApprovalThe present study was approved by the Institutional Ethics Committee of Osaka University Hospital (approved number: 13266-15). Animal studies were approved by the Institutional Animal Care and Use Committee (approved number: S20192D, S20197D and S20198D).

A Paper-based Immunofluorescent Device for the Detection of SARS-CoV-2 Humanized Antibody

We report on an immunofluorescent paper-based assay for the detection of severe acute respiratory symptom coronavirus 2 (SARS-CoV-2) humanized antibody. The paper-based device was fabricated by using lamination technique for easy and optimized handling. Our approach utilises a two-step strategy that involves (i) initial coating of the paper-electrode with recombinant SARS-CoV-2 nucleocapsid antigen to capture the target SARS-CoV-2 specific antibodies, and (ii) subsequent detection of SARS-CoV-2 antibodies using fluorophore-conjugated IgG antibody. The fluorescence readout was observed with fluorescence microscopy. The images were processed and quantified using a MATLAB program. The assay can selectively detect SARS-CoV-2 humanized antibodies spiked in PBS and healthy human serum samples with the relative standard deviation of approximately 6.4% (for n = 3). It has broad dynamic ranges (1 ng to 50 ng/µL in PBS and 5 to 100 ng/µL in human serum samples) for SARS-CoV-2 humanized antibodies with the detection limits of 2 ng/µL (0.025 IU/mL) and 10 ng/µL (0.125 IU/mL) in PBS and human serum samples, respectively. We believe that our assay has the potential to be used as a simple, rapid, and inexpensive paper-based diagnostic device with a portable fluorescent reader to provide point-of-care diagnosis. This assay can be used for rapid examination of a large batch of samples toward clinical screening of SARS-CoV-2 specific antibodies as a confirmed infected active case or to evaluate the immune response to a SARS-CoV-2 vaccine.

Antibody Humanization: Intra and Inter VH-VL Non-bond Energy Sort Best Humanized Antibody

Antibody humanization of non-human derived antibody reduces immunogenicity of antibody drug. Computer aided antibody humanization has became an efficient and rapid routine process. Our computational humanization pipeline includes CDR grafting onto antibody crystal structure and humanization of CDR grafted antibody structure. Then, intra and inter VH-VL non-bond energy is calculated and sorted for selection best humanized antibody. Compare to experimental dataset, result indicate that intra and inter VH-VL non-bond energy could rank humanized antibody.

PRECLINICAL STUDIES TO ASSESS ACUTE AND CHRONIC TOXICITIES OF A CANDIDATE DRUG BASED ON COMPLEMENT C3 NEODETERMINANT RECOMBINANT HUMANIZED ANTIBODY FOR TREATMENT OF TRAUMATIC BRAIN INJURIES

A drug based on complement C3 neodeterminant recombinant humanized antibody for the treatment of traumatic brain injuries belongs to the class III of low-risk drugs.