Engineering novel selectivity into metalloantibodies and targeting PcrV

Antibodies are a class of proteins that are secreted from our immune system to neutralize pathogens. They are very specific to the target cells and stably folded therefore they have become engaging for many scientists for decades. We were interested to study a novel group of antibodies that interacts to metals. Our findings indicated that metals are effective on stabilizing the antigen: antibody interaction. Previously, we were able to crystallize LT1009 antibody which is a humanized version of murine LT1002 IgG antibody. The crystal structure indicated that LT1009 employs two bridging calcium ions in the binding site to antigen. The biologically active lipid sphingosine-1-phosphate (S1P) was the antigen. Flame atomic absorption spectroscopy (FAAS) confirmed that murine LT1002 also binds calcium in solution and inductively coupled plasma-mass spectrometry (ICP-MS) indicates that, although calcium is the preferred metal binding cofactor, LT1002 can also bind to magnesium and, to a much lesser extent, barium. Isothermal titration calorimetry revealed that LT1002 binds two calcium ions endothermically with dissociation constants in the high μM range. Finally, an engineered, recombinantly expressed and purified antibody Fab fragment consisting of the murine germ-line encoded light and heavy chain genes from which LT1002 is likely derived and exhibits calcium binding profile as the mature antibody.Furthermore, we were interested to apply other metal bindings to the precursor metalloantibody. Therefore, we designed a large scale expression method to express the precursor antibody in Sf9 cells. Then, we were interested to design a plasmid by using site directed mutagenesis to change the key residues that were involved with the binding sites of Ca2+ and antigen. After that we were able to express and purify mutant antibody but this time the new metalloantibody binds to Zn2+. Engineered zinc antibodies can have the potential for site specific conjugation. Moreover, Zn2+ can protect cysteine from oxidation at the binding site. We propose that LT1002 is representative of a class of naturally occurring metalloantibodies that are evolutionarily conserved within the genomes of mammalian species

Determination of Binding Sites of Cadherin Peptides on the EC1 domain of E-cadherin using NMR Spectroscopy

The objective of this work is to evaluate the binding mechanisms of synthetic cadherin peptides (ADTC7, ADTC9 and cHAVc3) to the EC1 domain of human E-cadherin. The binding sites of these peptides to the EC1 domain were determined by titrating the 15N-labeled EC1 domain with each peptide. The changes in the 1H,15N-HSQC NMR spectra were observed upon peptide titration. The AutoDock Vina molecular modeling program was also used to predict the binding sites of each cadherin peptide on the EC1 domain. NMR data confirmed that there are three potential binding sites of ADTC7 on the EC1 domain; these binding sites are around residues I53/V48, D103, and G115. The two potential binding sites of ADTC9 peptide are around I4 and I53/V48 residues of EC1. The NMR data showed that ADTC7 peptide has higher binding affinity to the EC1 domain than ADTC9 peptide. For cHAVc3, it binds selectively to at the D103 residue on EC1. AutoDock Vina studies confirm some of the binding sites found by NMR studies. In the future, a more detail study will be conducted to evaluate the effect of peptide binding on the dynamics properties of the EC1 domain

Engineering novel selectivity into metalloantibodies and targeting PcrV

Antibodies are a class of proteins that are secreted from our immune system to neutralize pathogens. They are very specific to the target cells and stably folded therefore they have become engaging for many scientists for decades. We were interested to study a novel group of antibodies that interacts to metals. Our findings indicated that metals are effective on stabilizing the antigen: antibody interaction. Previously, we were able to crystallize LT1009 antibody which is a humanized version of murine LT1002 IgG antibody. The crystal structure indicated that LT1009 employs two bridging calcium ions in the binding site to antigen. The biologically active lipid sphingosine-1-phosphate (S1P) was the antigen. Flame atomic absorption spectroscopy (FAAS) confirmed that murine LT1002 also binds calcium in solution and inductively coupled plasma-mass spectrometry (ICP-MS) indicates that, although calcium is the preferred metal binding cofactor, LT1002 can also bind to magnesium and, to a much lesser extent, barium. Isothermal titration calorimetry revealed that LT1002 binds two calcium ions endothermically with dissociation constants in the high μM range. Finally, an engineered, recombinantly expressed and purified antibody Fab fragment consisting of the murine germ-line encoded light and heavy chain genes from which LT1002 is likely derived and exhibits calcium binding profile as the mature antibody.Furthermore, we were interested to apply other metal bindings to the precursor metalloantibody. Therefore, we designed a large scale expression method to express the precursor antibody in Sf9 cells. Then, we were interested to design a plasmid by using site directed mutagenesis to change the key residues that were involved with the binding sites of Ca2+ and antigen. After that we were able to express and purify mutant antibody but this time the new metalloantibody binds to Zn2+. Engineered zinc antibodies can have the potential for site specific conjugation. Moreover, Zn2+ can protect cysteine from oxidation at the binding site. We propose that LT1002 is representative of a class of naturally occurring metalloantibodies that are evolutionarily conserved within the genomes of mammalian species

Identification of novel anti-CD16a antibody clones for the development of effective natural killer cell engagers

Natural killer (NK) cells are key players in human innate immunity. Cell engager antibody formats that recruit and activate NK cells more effectively have emerged as a promising immunotherapy approach to target cancer cells through more effective antibody-dependent cell-mediated cytotoxicity (ADCC). Monoclonal antibody drugs with ADCC activity have shown clinical benefit and improved outcomes for patients with certain types of cancer. CD16a, a Fc gamma III receptor, is the major component that is responsible for the ADCC activity of NK cells. Screening AvantGen’s yeast displayed human antibody libraries led to the isolation of 2 antibody clones, #1A2 and #2-2A2, that selectively recognize both isoforms (F and V) of CD16a on primary NK cells with high affinity, yet minimally (#1A2) or do not (#2-2A2) cross-react with both allelotypes of CD16b (NA1 and NA2) expressed by neutrophils. Epitope mapping studies revealed that they bind to an epitope dependent on residue Y158 of CD16a, since mutation of Y158 to the corresponding CD16b residue H158 completely abolishes binding to CD16a. When formatted as bispecific antibodies targeting CD16a and a tumor-associated antigen (TAA, e.g. CD19), they exhibit specific binding to NK cells and induce potent NK cell activation upon encountering tumor cells, resulting in effective tumor cell killing. Notably, these bispecific antibody engagers stimulate NK cell cytokine release during co-culture with target cells, resulting in target cell cytotoxicity. These anti-CD16a antibody clones are promising candidates for combination with any TAA of interest, offering the potential for novel NK cell engager-based cancer therapeutics that are minimally affected by the high concentrations of human IgG in the circulation

Complementary antibody lineages achieve neutralization breadth in an HIV-1 infected elite neutralizer.

Broadly neutralizing antibodies (bNAbs) have remarkable breadth and potency against most HIV-1 subtypes and are able to prevent HIV-1 infection in animal models. However, bNAbs are extremely difficult to induce by vaccination. Defining the developmental pathways towards neutralization breadth can assist in the design of strategies to elicit protective bNAb responses by vaccination. Here, HIV-1 envelope glycoproteins (Env)-specific IgG+ B cells were isolated at various time points post infection from an HIV-1 infected elite neutralizer to obtain monoclonal antibodies (mAbs). Multiple antibody lineages were isolated targeting distinct epitopes on Env, including the gp120-gp41 interface, CD4-binding site, silent face and V3 region. The mAbs each neutralized a diverse set of HIV-1 strains from different clades indicating that the patient's remarkable serum breadth and potency might have been the result of a polyclonal mixture rather than a single bNAb lineage. High-resolution cryo-electron microscopy structures of the neutralizing mAbs (NAbs) in complex with an Env trimer generated from the same individual revealed that the NAbs used multiple strategies to neutralize the virus; blocking the receptor binding site, binding to HIV-1 Env N-linked glycans, and disassembly of the trimer. These results show that diverse NAbs can complement each other to achieve a broad and potent neutralizing serum response in HIV-1 infected individuals. Hence, the induction of combinations of moderately broad NAbs might be a viable vaccine strategy to protect against a wide range of circulating HIV-1 viruses

Identification of IOMA-class neutralizing antibodies targeting the CD4-binding site on the HIV-1 envelope glycoprotein

A major goal of current HIV-1 vaccine design efforts is to induce broadly neutralizing antibodies (bNAbs). The VH1-2-derived bNAb IOMA directed to the CD4-binding site of the HIV-1 envelope glycoprotein is of interest because, unlike the better-known VH1-2-derived VRC01-class bNAbs, it does not require a rare short light chain complementarity-determining region 3 (CDRL3). Here, we describe three IOMA-class NAbs, ACS101-103, with up to 37% breadth, that share many characteristics with IOMA, including an average-length CDRL3. Cryo-electron microscopy revealed that ACS101 shares interactions with those observed with other VH1-2 and VH1-46-class bNAbs, but exhibits a unique binding mode to residues in loop D. Analysis of longitudinal sequences from the patient suggests that a transmitter/founder-virus lacking the N276 glycan might have initiated the development of these NAbs. Together these data strengthen the rationale for germline-targeting vaccination strategies to induce IOMA-class bNAbs and provide a wealth of sequence and structural information to support such strategies