Discovery and optimization of a novel anti-GUCY2c x CD3 bispecific antibody for the treatment of solid tumors.

We report here the discovery and optimization of a novel T cell retargeting anti-GUCY2C x anti-CD3ε bispecific antibody for the treatment of solid tumors. Using a combination of hybridoma, phage display and rational design protein engineering, we have developed a fully humanized and manufacturable CD3 bispecific antibody that demonstrates favorable pharmacokinetic properties and potent in vivo efficacy. Anti-GUCY2C and anti-CD3ε antibodies derived from mouse hybridomas were first humanized into well-behaved human variable region frameworks with full retention of binding and T-cell mediated cytotoxic activity. To address potential manufacturability concerns, multiple approaches were taken in parallel to optimize and de-risk the two antibody variable regions. These approaches included structure-guided rational mutagenesis and phage display-based optimization, focusing on improving stability, reducing polyreactivity and self-association potential, removing chemical liabilities and proteolytic cleavage sites, and de-risking immunogenicity. Employing rapid library construction methods as well as automated phage display and high-throughput protein production workflows enabled efficient generation of an optimized bispecific antibody with desirable manufacturability properties, high stability, and low nonspecific binding. Proteolytic cleavage and deamidation in complementarity-determining regions were also successfully addressed. Collectively, these improvements translated to a molecule with potent single-agent in vivo efficacy in a tumor cell line adoptive transfer model and a cynomolgus monkey pharmacokinetic profile (half-life>4.5 days) suitable for clinical development. Clinical evaluation of PF-07062119 is ongoing.

Sperm-Coating Beta-Defensin 126 Is a Dissociation-Resistant Dimer Produced by Epididymal Epithelium in the Bovine Reproductive Tract.

Beta-defensins are innate immune molecules, often described as antimicrobial peptides because of their bactericidal activity and are now known to have diverse additional functions, including cell signaling, chemoattraction, immunoregulation, and reproduction. In humans and primates, beta-defensin 126 has been shown to regulate the ability of sperm to swim through cervical mucus and to protect sperm from attack by the female immune system during transit toward the oviduct. Bovine beta-defensin 126 (BBD126) is the ortholog of human defensin 126, and computational analysis here revealed significant conservation between BBD126 and other mammalian orthologs at the N-terminus, although extensive sequence differences were detected at the C-terminus, implying possible species-specific roles for this beta-defensin in reproduction. We had previously demonstrated preferential expression of this and related beta-defensin genes in the bovine male reproductive tract, but no studies of bovine beta-defensin proteins have been performed to date. Here, we analyzed BBD126 protein using a monoclonal antibody (a-BBD126) generated against a 14 amino acid peptide sequence from the secreted fragment of BBD126. The specificity of a-BBD126 was validated by testing against the native form of the peptide recovered from bovine caudal epididymal fluid and recombinant BBD126 generated using a prokaryotic expression system. Western blot analysis of the native and recombinant forms showed that BBD126 exists as a dimer that was highly resistant to standard methods of dissociation. Immunohistochemical staining using a-BBD126 demonstrated BBD126 protein expression by epithelial cells of the caudal epididymis and vas deferens from both mature and immature bulls. BBD126 could also be seen (by confocal microscopy) to coat caudal sperm, with staining concentrated on the tail of the sperm cells. This study is the first to demonstrate beta-defensin 126 protein expression in the bovine reproductive tract and on bull sperm. Its dissociation-resistant dimeric structure is likely to have important functional implications for the role of BBD126 in bovine reproduction.

Cauda Epididymis-Specific Beta-Defensin 126 Promotes Sperm Motility but Not Fertilizing Ability in Cattle.

Bovine beta-defensin 126 (BBD126) exhibits preferential expression for the cauda epididymis of males, where it is absorbed onto the tail and postacrosomal region of the sperm. The aim of this study was to examine the role of BBD126 in bull sperm function. Fresh and frozen-thawed semen were incubated in the presence of different capacitating agents as well as with phosphatidylinositol-specific phospholipase C. These treatments, which have been successful in releasing beta-defensin 126 from macaque sperm, proved to be ineffective in bull sperm. This finding suggests that the protein behaves in a different manner in the bovine. The lack of success in removing BBD126 led us to use corpus epididymis sperm, a model in which the protein is not present, to study its functional role. Corpus sperm were incubated with cauda epididymal fluid (CEF) in the absence or presence of BBD126 antibody or with recombinant BBD126 (rBBD126). Confocal microscopy revealed that rBBD126 binds to corpus sperm with the same pattern observed for BBD126 in cauda sperm, whereas an aberrant binding pattern is observed when sperm are subject to CEF incubation. Addition of CEF increased motility as well as the number of corpus sperm migrating through cervical mucus from estrus cows. However, it decreased the ability of sperm to fertilize in vitro matured oocytes. The presence of the antibody failed to abrogate these effects. Furthermore, when rBBD126 was added in the absence of other factors and proteins from the CEF, an increase in motility was also observed and no negative effects in fertility were seen. These results suggest that BBD126 plays a key role in the acquisition of sperm motility in the epididymis.

Characterisation and expression profile of the bovine cathelicidin gene repertoire in mammary tissue.

BACKGROUND: Cathelicidins comprise a major group of host-defence peptides. Conserved across a wide range of species, they have several functions related to host defence. Only one cathelicidin has been found in humans but several cathelicidin genes occur in the bovine genome. We propose that these molecules may have a protective role against mastitis. The aim of this study was to characterise the cathelicidin gene-cluster in the bovine genome and to identify sites of expression in the bovine mammary gland. RESULTS: Bioinformatic analysis of the bovine genome (BosTau7) revealed seven protein-coding cathelicidin genes, CATHL1-7, including two identical copies of CATHL4, as well as three additional putative cathelicidin genes, all clustered on the long arm of chromosome 22. Six of the seven protein-coding genes were expressed in leukocytes extracted from milk of high somatic cell count (SCC) cows. CATHL5 was expressed across several sites in the mammary gland, but did not increase in response to Staphylococcus aureus infection. CONCLUSIONS: Here, we characterise the bovine cathelicidin gene cluster and reconcile inconsistencies in the datasets of previous studies. Constitutive cathelicidin expression in the mammary gland suggests a possible role for these host defence peptides its protection.

The postpartum endometrial inflammatory response: a normal physiological event with potential implications for bovine fertility.

After calving, the bovine endometrium undergoes marked morphological and functional changes that are necessary for subsequent re-breeding. Regulation and integration of these key events are largely uncharacterised. Here, endometrial swabs and biopsies were taken at 15, 30 and 60 days postpartum (DPP) from 13 healthy primiparous cows, 10 of which subsequently conceived, with a view to characterising innate and inflammatory gene expression profiles. Endometrial biopsies exhibited severe inflammation (>75 leukocytes per high-power field) at 15 DPP, which had begun to resolve by 30 DPP and had completely resolved by 60 DPP. The severe inflammation at 15 DPP coincided with uterine infection in all cows and a significant increase (P < 0.05) in the expression of all of 16 genes investigated, including CD45, IL8, IL6, IL1, TNF, TAP, SAA3 and HP at 15 DPP, relative to 60 DPP. All of these parameters had begun to return to normal physiological levels at 30 DPP. Systemically, serum protein concentrations of IL-8 were elevated at 15 DPP compared with 60 DPP (78 pgmL(-1)vs 48 pgmL(-1); P = 0.02). These results indicate that endometrial inflammation, leukocyte infiltration and increased expression of pro-inflammatory, antimicrobial and acute-phase protein genes are expected features of the postpartum period, critical to bacterial clearance and uterine involution.

Global endometrial transcriptomic profiling: transient immune activation precedes tissue proliferation and repair in healthy beef cows.

BACKGROUND: All cows experience bacterial contamination and tissue injury in the uterus postpartum, instigating a local inflammatory immune response. However mechanisms that control inflammation and achieve a physiologically functioning endometrium, while avoiding disease in the postpartum cow are not succinctly defined. This study aimed to identify novel candidate genes indicative of inflammation resolution during involution in healthy beef cows. Previous histological analysis of the endometrium revealed elevated inflammation 15 days postpartum (DPP) which was significantly decreased by 30 DPP. The current study generated a genome-wide transcriptomic profile of endometrial biopsies from these cows at both time points using mRNA-Seq. The pathway analysis tool GoSeq identified KEGG pathways enriched by significantly differentially expressed genes at both time points. Novel candidate genes associated with inflammatory resolution were subsequently validated in additional postpartum animals using quantitative real-time PCR (qRT-PCR). RESULTS: mRNA-Seq revealed 1,107 significantly differentially expressed genes, 73 of which were increased 15 DPP and 1,034 were increased 30 DPP. Early postpartum, enriched immune pathways (adjusted P < 0.1) included the T cell receptor signalling pathway, graft-versus-host disease and cytokine-cytokine receptor interaction pathways. However 30 DPP, where the majority of genes were differentially expressed, the enrichment (adjusted P < 0.1) of tissue repair and proliferative activity pathways was observed. Nineteen candidate genes selected from mRNA-Seq results, were independently assessed by qRT-PCR in additional postpartum cows (5 animals) at both time points. SAA1/2, GATA2, IGF1, SHC2, and SERPINA14 genes were significantly elevated 30 DPP and are functionally associated with tissue repair and the restoration of uterine homeostasis postpartum. CONCLUSIONS: The results of this study reveal an early activation of the immune response which undergoes a temporal functional change toward tissue proliferation and regeneration during endometrial involution in healthy postpartum cows. These molecular changes mirror the activation and resolution of endometrial inflammation during involution previously classified by the degree of neutrophil infiltration. SAA1/2, GATA2, IGF1, SHC2, and SERPINA14 genes may become potential markers for resolution of endometrial inflammation in the postpartum cow.

Functional characterisation of bovine interleukin 8 promoter haplotypes in vitro.

Interleukin 8 (IL-8) is a major mediator of the innate immune response and polymorphisms in this gene are associated with susceptibility to inflammatory disease in humans. The aim of this study was to characterise the promoter region of the bovine IL8 gene towards understanding its regulation and the effect of promoter polymorphisms on gene expression levels. Twenty-nine polymorphic sites were identified across a 2.1kb upstream promoter region of the IL8 gene including two insertion/deletion polymorphisms. Sequence analysis and SNP genotyping identified two distinct promoter haplotypes (IL8-h1 and IL8-h2), which were present at significantly different frequencies in two divergently selected cattle breeds - Holstein-Friesian and Norwegian Red (IL8-h1 at 48% and 80% respectively). IL8-h1 was functionally less responsive in unstimulated mammary epithelial cells and in response to stimulation with LPS or bovine TNF. Serial deletion analysis and in silico transcription-factor binding site analysis indicated that allele specific binding of the transcriptional repressor Oct-1 may account for the reduced sensitivity of IL8-h1. Our finding of genetic variation in the bovine IL8 promoter that differentially regulates its expression has significant functional implications for IL8 expression in vitro and which may impact on susceptibility to bovine infectious disease and inflammation.

Reproductive tissue-specific expression profiling and genetic variation across a 19 gene bovine ß-defensin cluster.

ß-defensins are small cationic peptides, with potent immunoregulatory and antimicrobial activity which are produced constitutively and inducibly by eukaryotic cells. This study profiles the expression of a cluster of 19 novel defensin genes which spans 320 kb on chromosome 13 in Bos taurus. It also assesses the genetic variation in these genes between two divergently selected cattle breeds. Using quantitative real-time PCR (qRT-PCR), all 19 genes in this cluster were shown to be expressed in the male genital tract and 9 in the female genital tract, in a region-specific manner. These genes were sequenced in Norwegian Red (NR) and Holstein-Friesian (HF) cattle for population genetic analysis. Of the 17 novel single nucleotide polymorphisms (SNPs) identified, 7 were non-synonymous, 6 synonymous and 4 outside the protein coding region. Significant frequency differences in SNPs in bovine ß-defensins (BBD) 115, 117, 121, and 122 were detected between the two breeds, which was also reflected at the haplotype level (P<0.05). There was clear segregation of the haplotypes into two blocks on chromosome 13 in both breeds, presumably due to historical recombination. This study documents genetic variation in this ß-defensin gene cluster between Norwegian Red and Holstein-Friesian cattle which may result from divergent selection for production and fertility traits in these two breeds. Regional expression in the epididymis and fallopian tube suggests a potential reproductive-immunobiology role for these genes in cattle.

Divergent antimicrobial peptide (AMP) and acute phase protein (APP) responses to Trypanosoma congolense infection in trypanotolerant and trypanosusceptible cattle.

African animal trypanosomiasis (AAT) is endemic across Sub-Saharan African and is a major constraint to livestock production. The ability of certain cattle breeds to remain productive despite infection is known as trypanotolerance; however, the underlying immune mechanisms contributing to this trait remain poorly understood. Antimicrobial peptides (AMPs) and acute phase proteins (APPs) are evolutionarily conserved effector molecules of the innate immune system that have important roles in the resolution of infection and activation of the adaptive immune response. Expression levels of AMP genes (TAP, LAP, BNBD4, DEFB1, DEFB5 and LEAP2) and APP genes (HP, CP, AGP, LBP, SAA3 and CRP) were investigated using real time quantitative reverse transcription PCR (qRT-PCR) in peripheral blood mononuclear cells (PBMC) isolated from two breeds of African cattle (trypanotolerant N'Dama and trypanosusceptible Boran), experimentally infected with Trypanosoma congolense. Haptoglobin and serum amyloid A (SAA) were also measured in plasma using quantitative protein assays. Results demonstrated that tracheal antimicrobial peptide (TAP) gene expression increased by 32-fold in Boran, compared to only 3-fold in N'Dama, by 14 days post-infection (dpi) and rising to 136-fold at 29 dpi in Boran, compared to 47-fold in N'Dama (P<0.05). Protein expression levels of SAA are elevated in N'Dama, rising to 163 microg/ml at 14 dpi compared with 72 microg/ml in Boran. The SAA expression profile mirrors the wave of parasitaemia detected in N'Dama. Seven single nucleotide polymorphisms (SNPs) were identified in the promoter regions of the SAA3 and SAA4 genes, which are predicted to affect transcription factor binding and thereby contributing to the differential patterns of expression detected between the breeds. Whereas elevated TAP expression is a conserved component of the innate immune response to infection in both breeds, higher SAA expression levels may contribute toward trypanotolerance in N'Dama.

Comparative in vivo infection models yield insights on early host immune response to Campylobacter in chickens.

Salmonella typhimurium and Campylobacter jejuni pose significant risks to human health and poultry are a major vector for infection. Comparative in vivo infection models were performed to compare the avian host immune response to both bacterial species. Forty-five commercial broiler chickens were orally challenged with either C. jejuni or S. typhimurium whilst 60 similar control birds were mock challenged in parallel. Birds were sacrificed at 0, 6, 20 and 48 h post-infection and cloacal swabs, blood and tissue samples taken. Peripheral blood leukocytes were isolated for flow cytometric analyses and RNA was extracted for gene expression profiling. Colonisation patterns were markedly different between the two bacterial species, with systemic colonisation of Campylobacter outside the gastrointestinal tract. Salmonella infection induced significant changes in circulating heterophil and monocyte/macrophage populations, whilst Campylobacter infection had no effect on the heterophil numbers but caused a significant early increase in circulating monocytes/macrophages. Toll-like receptor 1 (TLR1) gene expression was decreased, and avian beta-defensin (AvBD) gene expression (AvBD3, AvBD10 and AvBD12) was significantly increased in response to Salmonella infection (P < 0.05). In contrast, Campylobacter infection induced increased TLR21 gene expression but significantly reduced expression of seven antimicrobial peptide (AMP) genes (AvBD3, AvBD4, AvBD8, AvBD13, AvBD14, CTHL2 and CTHL3; P < 0.05). Considered together, microbiological, cellular and gene expression profiles indicate that the innate immune system responds differently to Salmonella and to Campylobacter infection. Furthermore, reduction in the expression of AMPs may play a role in the persistence of high level colonisation of the host by Campylobacter.

Directed alteration of a novel bovine beta-defensin to improve antimicrobial efficacy against methicillin-resistant Staphylococcus aureus (MRSA).

beta-Defensin antimicrobial peptides are critical components of the innate immune response in many species and may be useful against pathogens that have acquired resistance to standard antibiotic therapies. We analysed a panel of recently discovered bovine beta-defensins in order to identify sites that may have particular functional importance against antibiotic-resistant bacteria. Modifications were introduced to increase charge at positively selected (PS) sites, to make charge-neutral changes at PS sites, to increase hydrophobicity and to confer a hydrophilic C-terminal. Whilst all four peptide modifications increased antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) compared with the native form of bovine beta-defensin 123 (P=0.02), conferring the hydrophilic tail caused the most significant increase, with a 50% lethal dose (LD(50)) of 3.91 microg/mL. The peptide with increased charge at PS sites showed the most significant increase in antimicrobial activity against a non-resistant strain of S. aureus (P=0.02). Therefore, informed modifications of the amino acid sequence can significantly affect the specificity and antimicrobial activity of a peptide.

Evolution, expression and effectiveness in a cluster of novel bovine beta-defensins.

The anti-microbial peptides beta-defensins constitute a large family of innate immune effector molecules, conserved across a wide species range. In this paper, we describe a systematic search of the sequenced bovine genome to characterise this extensive gene family in Bos taurus, providing an insight into the pattern of conservation of beta-defensin genes between species. We have sequenced a sub-set of these newly discovered bovine beta-defensin genes and also report expression data for these genes across a range of tissues. We have synthesised the peptide product of one of these genes, bovine beta-defensin 123, and found it to be a potent inhibitor of several pathogenic microbes, particularly Escherichia coli and Listeria monocytogenes.