Antibodies targeting quiescin sulfhydryl oxidase (QSOX1) and uses of same
Cross-species-specific antibodies targeting QSOX1, with enhanced binding affinity through structural modifications, address species specificity issues, improving therapeutic potential for treating laminin-associated diseases by effectively inhibiting both human and mouse QSOX1.
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example 1
The QS0X1 Inhibitor MAb492.1 is Species-Specific
[0319]MAb492.1. which inhibits HsQSOX1 activity in vitro and in cell culture at a near-stoichiometric concentration [Grossman I. et al. (2013) supra], was tested on other mammalian QSOX1 enzymes to find a suitable model for assessing QSOX1 inhibition in vivo. Three mammalian QSOX1 enzymes, from common experimental organisms and having 78-79% sequence identity with HsQSOX1, were chosen: Mus musculus QSOX1 (MmQSOX1), Rattus norvegicus QSOX1 (RnQSOX1), and Cavia porcellus (CpQSOX1). Enzyme activity was assessed in an oxygen consumption assay on the model substrate dithiothreitol (DTT). According to the assay, MAb492.1 had no effect on MmQSOX1, RnQSOX1, or CpQSOX1, even at 1 μM, a 10:1 molar ratio of antibody to enzyme (FIG. 2A).
[0320]To understand the molecular basis for MAb492.1 species restriction, the epitope on HsQSOX1 was compared with the corresponding regions of RnQSOX1, CpQSOX1, and particularly MmQSOX1 by crystallizing and solvin...
example 2
Generation and Characterization of a Murine Antibody Inhibitor Targeting MmQSOX1
[0322]The species-specificity exhibited by MAb492.1 is a common feature shared by natural monoclonal antibodies. Although differences in antigen structure might be small among orthologs, they are exploited by the immune system to bind the foreign antigen and avoid self-reactivity. In the large antigen surface area buried by antibodies, minor differences between orthologs can cause steric clashes that cannot be remedied by a simple corresponding change in the antibody. QSOX1 knock-out (QSOX1.-KO) mice produced in the laboratory were then exploited to generate surrogate antibodies against MmQSOX1, which is a foreign antigen for these animals. Hybridoma supernatants were screened for binding of MmQSOX1 using a standard enzyme-linked immunosorbent assay (ELISA). Top hinders were tested for MmQSOX1 inhibition, and five were chosen for sub-cloning (described in the materials and experimental procedures section...
example 3
Generation of a MAb492.1 Variant Targeting MmQOX1
[0323]In parallel to obtaining an antibody targeting MmQSOX1 T from hybridoma clones, a variant of MAb492.1 that inhibits MmQSOX1 was developed. Though occasionally a single point mutation may modulate antibody species specificity to some extent, substantial re-engineering is often required to obtain the desired target-recognition properties. According to the observation that mutating four residues of MmQSOX1 was sufficient to achieve inhibition by MAb492.1 (FIG. 2D), inventors reasoned that inhibiting MmQSOX1 would be possible by making a set of mutations in the MAb492.1 CDRs or surrounding regions. However, in contrast to modifying MmQSOX1. to mimic HsQSOX1, modification of the antibody had to be made without a guiding structure. Furthermore, the four QSOX1 residues controlling reactivity with MAb492.1 affected the position of the QSOX1 polypeptide backbone (FIG. 2C), hinting that substantial compensating mutations affecting CDR loo...
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