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Imvotamab (IGM-2323) is an engineered high-affinity, high-avidity bispecific anti-CD20 IgM antibody T cell engager (TCE) that is currently being studied as monotherapy in a Phase 1/2 clinical trial for relapsed/refractory non-Hodgkin’s lymphoma (NHL) (NCT04082936). Imvotamab offers a novel treatment strategy in NHL by depleting CD20-expressing tumor cells through multiple mechanisms, including the recruitment of T cells to kill tumor cells through T cell dependent cellular cytotoxicity, complement-dependent cytotoxicity, and enhanced immune modulation via IFNγ-dominant cytokine stimulation. We evaluated the activity of a surrogate cynomolgus monkey cross-reactive CD20xCD3 IgM bispecific TCE, IGM-2324, in depleting CD20-expressing B cells in peripheral blood and lymphoid tissues of cynomolgus monkeys in vivo. We hypothesized that the high affinity and valency of IGM-2324 would enable potent B cell killing in blood and tissues even when B cells express low levels of CD20. Cynomolgus monkeys were administered vehicle or IGM-2324 at 5 mg/kg or 25 mg/kg through intravenous infusion twice weekly for a total of four doses on days 1, 4, 7, and 10. B cell depletion in peripheral blood was assessed by measuring the frequency of CD19+ B cells through flow cytometry. Administration of IGM-2324 at 5 and 25 mg/kg resulted in a nearly complete depletion in peripheral CD19+ B cells at 8 hours post the 1st dose through 24 hours post the last dose on day 11. Depletion of tissue-resident B cells was evaluated in the spleen, mesenteric lymph node (MLN) and bone marrow (BM) of monkeys at 24 hours post the last dose of vehicle or IGM-2324 on day 11. Immunohistochemistry studies were conducted on the formalin-fixed paraffin-embedded lymphoid tissues by staining for CD19 and CD20 expression. The number and intensity of CD19 or CD20 positive B cells were determined by quantitative imaging analysis. Compared to vehicle-treated animals, significant dose-dependent reductions in both CD19 and CD20-expressing B cells were observed in spleen, MLN and BM following treatment with 5 and 25 mg/kg of IGM-2324. Most importantly, IGM-2324 treatment led to the depletion of not only high and moderate tissue-resident CD20-expressing B cells, but also B cells that expressed low levels of CD20. Our preclinical data indicate that a CD20xCD3 IgM bispecific TCE can penetrate tissues and mediate direct killing of CD20-expressing target cells. B cell depletion in the periphery and tumors of relapsed/refractory NHL patients is currently being evaluated as biomarker of pharmacodynamic activity and/or efficacy for imvotamab in a Phase 1/2 clinical study. Citation Format: Miho Oyasu, Angus M. Sinclair, Haben Ghermazien, Genevive Hernandez, Thomas Manley, Maya K. Leabman, Stephen F. Carroll, Bruce A. Keyt, Maya F. Kotturi. Depletion of tissue-resident B cells by a CD20xCD3 IgM bispecific T cell engager in cynomolgus monkeys demonstrates effective tissue penetration and potent target cell killing. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4120.

Rituximab-containing treatment regimens are the standard of care for patients with non-Hodgkin’s lymphoma (NHL). However, the majority of patients ultimately experience disease relapse or progression indicating resistance to rituximab therapy. IGM-2323 is an engineered high-affinity, high-avidity anti-CD20 pentameric IgM antibody with an anti-CD3 scFv fused to the joining chain. IGM-2323 offers a novel treatment strategy in NHL through two mechanisms: 1) the recruitment of T cells to kill CD20-expressing tumor cells through T cell dependent cellular cytotoxicity (TDCC) and, 2) complement-dependent cytotoxicity (CDC). We evaluated the activity of IGM-2323 in the presence of rituximab since rituximab can persist in patients after treatment discontinuation, and it can bind to an overlapping epitope on CD20 as IGM-2323. We hypothesized that the high valency of IGM-2323 could displace rituximab, thus enabling potent B cell killing by IGM-2323 even in the presence of high concentrations of rituximab. The affinity of IGM-2323 and its corresponding bivalent anti-CD20 IgG antibody to recombinant human CD20 protein were measured by surface plasmon resonance. IGM-2323 bound to human CD20 with an apparent 300-fold higher binding affinity (KD) and ~100-fold slower off-rate (kdis) than the bivalent anti-CD20 IgG. Human B cell lines with a range of CD20 expression levels, including a CD20-low rituximab-resistant Ramos cell variant, were pre-treated with escalating concentrations of rituximab, and subsequently evaluated in vitro for cell binding, TDCC, and CDC by IGM-2323. At high concentrations of rituximab, which correspond to reported peak serum concentrations (Cmax) found in rituximab-treated patients, IGM-2323 displaced the binding of rituximab on human B cell lines. In contrast, binding of a bispecific CD20xCD3 IgG was severely inhibited by the Cmax of rituximab. In TDCC assays with healthy donor effector T cells, pre-treatment with high concentrations of rituximab only modestly inhibited IGM-2323 activity. Furthermore, only a minor impact to the maximum killing activity (Emax) of IGM-2323 was observed at the Cmax of rituximab. In contrast, rituximab pre-treatment resulted in a distinctly lower Emax of a bispecific CD20xCD3 IgG. Live cell imaging of CDC kinetics was utilized to quantify the extent of CDC by IGM-2323 with or without rituximab. Pre-treatment with rituximab enhanced CDC of IGM-2323 compared to single agent activity. Our preclinical data indicate that IGM-2323 maintains activity in the presence of rituximab. IGM-2323 is currently being studied in a phase 1 clinical trial in relapsed/refractory NHL, where it has been generally well tolerated, with both complete and partial responses observed (NCT04082936). Clinical studies will continue to evaluate these findings, including the treatment of patients with circulating serum levels of rituximab. Citation Format: Kevin C. Hart, Kathryn Logronio, Mandy Li, Poonam Yakkundi, Marigold Manlusoc, Keyu Li, Paul R. Hinton, Dean Ng, Maya K. Leabman, Genevive Hernandez, Thomas Manley, Angus M. Sinclair, Stephen F. Carroll, Bruce A. Keyt, Maya F. Kotturi. High valency of IGM-2323, a CD20xCD3 IgM bispecific T cell engager, displaces rituximab binding and induces potent B lymphoma cell killing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4179.

ObjectiveThe goal of this study was to determine the effect of a genetic variant in the organic cation transporter 2 (OCT2), OCT2-808G/T, which results in an amino acid change, A270S, on the pharmacokinetics of the antidiabetic drug, metformin.MethodsThe uptake of metformin was performed in stably t

The pH-dependent binding of Igs to the neonatal FcR (FcRn) plays a critical role in the in vivo homeostasis of IgGs. Modulating the interaction between Fc and FcRn through protein engineering is one method for improving the pharmacokinetics of therapeutic Abs. Recent studies disputed the direct relationship between increasing FcRn affinity and improved pharmacokinetic properties. In this work, we studied the pharmacokinetics of two human IgG1 Fc variants in cynomolgus monkey to further clarify the affinity-pharmacokinetic relationship. First, we report a number of novel Fc point mutations and combination variants, including some with primate-specific FcRn-binding improvements. By studying these variants along with some previously described variants across a wide range of affinities, we discovered a direct correlation of pH 6 affinity improvements with neutral pH improvements, suggesting that all of the tested variants exhibit similar pH dependency in FcRn binding. We then evaluated the pharmacokinetics of variants N434A and N434W, which, respectively, gave ∼4- and 80-fold improvements in pH 6-binding affinity to both human and nonhuman primate FcRn. Surprisingly, clearance of N434W was similar to that of wild type. N434W is the first variant studied in primates that exhibits significant binding to FcRn at pH 7.4, and its clearance substantiates the principle that too much affinity improvement, i.e., beyond that of N434W, does not yield improved pharmacokinetics. In contrast, N434A exhibited a ∼2-fold decrease in clearance in cynomolgus monkey, supporting the notion that modest increases in pH 6 FcRn affinity can result in improved pharmacokinetics in primates.

The human organic cation transporter 2 (OCT2, SLC22A2 ) is a multispecific transporter of organic cations, including many clinically used drugs. OCT2 is primarily responsible for the uptake of organic cations across the basolateral membrane of renal tubular epithelial cells and is considered a major transporter in the active secretion of organic cations in the kidney. Uptake of organic cations by OCT2 is driven by the inside-negative membrane potential and is pH-sensitive. Regulation of OCT2 at the transcriptional level by steroid hormones and at the protein level by various protein kinases has been described. Several human genetic variants in the coding region of OCT2 have been identified and functionally characterized, including both polymorphic and rare variants. A variety of structurally diverse compounds have been shown to interact with OCT2, including endogenous compounds, drugs, and dietary supplements. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association

Membrane transporters maintain cellular and organismal homeostasis by importing nutrients and exporting toxic compounds. Transporters also play a crucial role in drug response, serving as drug targets and setting drug levels. As part of a pharmacogenetics project, we screened exons and flanking intronic regions for variation in a set of 24 membrane transporter genes (96 kb; 57% coding) in 247 DNA samples from ethnically diverse populations. We identified 680 single nucleotide polymorphisms (SNPs), of which 175 were synonymous and 155 caused amino acid changes, and 29 small insertions and deletions. Amino acid diversity (π NS ) in transmembrane domains (TMDs) was significantly lower than in loop domains, suggesting that TMDs have special functional constraints. This difference was especially striking in the ATP-binding cassette superfamily and did not parallel evolutionary conservation: there was little variation in the TMDs, even in evolutionarily unconserved residues. We used allele frequency distribution to evaluate different scoring systems (Grantham, blosum 62, SIFT, and evolutionarily conserved/evolutionarily unconserved) for their ability to predict which SNPs affect function. Our underlying assumption was that alleles that are functionally deleterious will be selected against and thus under represented at high frequencies and over represented at low frequencies. We found that evolutionary conservation of orthologous sequences, as assessed by evolutionarily conserved/evolutionarily unconserved and SIFT, was the best predictor of allele frequency distribution and hence of function. European Americans had an excess of high frequency alleles in comparison to African Americans, consistent with a historic bottleneck. In addition, African Americans exhibited a much higher frequency of population specific medium-frequency alleles than did European Americans.

The completion of the Human Genome Project and the development of high-throughput polymorphism identification methods have allowed researchers to carry out full genetic analyses of many clinically relevant genes. However, few studies have combined genetic analysis with in vitro phenotyping to better understand the relationship between genetic variation and protein function. Many transporters in the kidney are thought to play key roles in defense against a variety of foreign substances. The goal of this study was to understand the relationship between variation in a gene encoding a major renal xenobiotic transporter, OCT2, and transporter function. We report a comprehensive genetic analysis and functional characterization of variants of OCT2. Twenty-eight variable sites in the OCT2 gene were identified in a collection of 247 ethnically diverse DNA samples. Eight caused non-synonymous amino acid changes, of which four were present at/= 1% in an ethnic population. All four of these altered transporter function assayed in Xenopus laevis oocytes. Analysis of nucleotide diversity (pi) revealed a higher prevalence of synonymous (pi = 22.4 x 10-4) versus non-synonymous (pi = 2.1 x 10-4) changes in OCT2 than in other genes. In addition, the non-synonymous sites had a significant tendency to exhibit more skewed allele frequencies (more negative Tajima's D-values) compared to synonymous sites. The population-genetic analysis, together with the functional characterization, suggests that selection has acted against amino acid changes in OCT2. This selection may be due to a necessary role of OCT2 in the renal elimination of endogenous amines or xenobiotics, including environmental toxins, neurotoxic amines and therapeutic drugs.

Transporters in the kidney mediate the secretion or reabsorption of many compounds and thereby influence the plasma levels of their substrates. Organic anion transporters and organic cation transporters are two major classes of secretory transporters in the mammalian kidney. During the past decade, significant progress has been made in the cloning, functional expression, and initial characterization of these transporters. To date, five organic cation transporters and nine organic anion transporters have been cloned. In this review, we summarize the available data on organic anion and organic cation transporters, focusing in particular on their molecular characteristics, tissue distribution, and inhibitor and substrate selectivities. Currently we have a good understanding of the inhibitor selectivities for most of these transporters, and with the development of more robust assays, we will soon have a better understanding of their substrate selectivities. Based on the available data, summarized in this review, it appears that many compounds interact with multiple transporters. Futhermore, there appears to be substantial overlap in the selectivities of organic cation transporters, and the same appears true for organic anion transporters. At the present time, it is unclear what the roles of these multiple transporters are in renal drug elimination. With the development of new assays, reagents, and experimental methods, we will soon have a better understanding of the roles of each transporter isoform in the renal elimination of drugs. © 2001 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:397–421, 2001

Category: genotype Project: Pharmacogenetics of Membrane Transporters [Table 1][1] provides HUGO Gene Nomenclature Committee (HGNC) symbols, PharmGKB submission URLs, submission dates, and release dates. [Table 2][2] provides HGNC symbols, HGNC names, synonyms, GenBank accession numbers, and locus

Although considerable progress has been made toward characterizing human DNA sequence variation, there remains a deficiency in information on human phenotypic variation at the single-gene level. We systematically analyzed the function of all protein-altering variants of eleven membrane transporters in heterologous expression systems. Coding-region variants were identified by screening DNA from a large sample (n = 247-276) of ethnically diverse subjects. In total, we functionally analyzed 88 protein-altering variants. Fourteen percent of the polymorphic variants (defined as variants with allele frequencies ≥1% in at least one major ethnic group) had no activity or significantly reduced function. Decreased function variants had significantly lower allele frequencies and were more likely to alter evolutionarily conserved amino acid residues. However, variants at evolutionarily conserved positions with approximately normal activity in cellular assays were also at significantly lower allele frequencies, suggesting that some variants with apparently normal activity in biochemical assays may influence occult functions or quantitative degrees of function that are important in human fitness but not measured in these assays. For example, eight (14%) of the 58 variants for which we had measured the transport of at least two substrates showed substrate-specific defects in transport. These variants and the reduced function variants provide plausible candidates for disease susceptibility or variation in clinical drug response.

The organic anion transporter, OAT1 (SLC22A6), plays a role in the renal elimination of many drugs and environmental toxins. The goal of this study was to identify and functionally characterize OAT1 variants as a first step towards understanding whether genetic variation in OAT1 may contribute to interindividual differences in renal elimination of xenobiotics.As part of a larger study, 276 DNA samples from an ethnically diverse population were screened and 12 coding region variants of OAT1 were identified. The non-synonymous variants were then constructed and characterized in Xenopus laevis oocytes. A small family-based clinical study was conducted to determine the renal elimination of a model OAT1 substrate, adefovir (an antiviral agent) in human subjects who possessed a non-functional variant, OAT1-R454Q.Six non-synonymous variants were identified; two (OAT1-R50 H and OAT1-R293W) were present ator = 1% in at least one ethnic population. These two variants exhibited normal uptake of p-aminohippurate, ochratoxin A and methotrexate assayed in X. laevis oocytes. One variant, OAT1-R454Q, was non-functional with respect to the above substrates. In the clinical study, there was no significant decrease in the renal secretory clearance of adefovir in family members heterozygous for OAT1-454Q in comparison to those with the reference transporter, OAT1-454R.These data indicate that the coding region of OAT1 has low genetic and functional diversity and suggest that coding region variants of OAT1 may not contribute substantially to interindividual differences in renal elimination of xenobiotics.

Category: Genotype PharmGKB Submission Number: PS203001 Date of Submission: June 16, 2003 Project: Pharmacogenetics of Membrane Transporters HGNC Symbol: SLC22A1 HGNC Name: solute carrier family 22 (organic cation transporter), member 1

Renal excretion is the major pathway for elimination of many clinically used drugs and xenobiotics. We estimated the genetic component (rGC) contributing to variation in renal clearance for six compounds (amoxicillin, ampicillin, metformin, terodiline, digoxin and iohexol) using Repeated Drug Application methodology. Data were obtained from published literature. The rGC values of renal clearance of metformin, amoxicillin, and ampicillin, which undergo transporter-mediated secretion, ranged from 0.64-0.94. This finding suggests that variation in the renal clearance of these drugs has a strong genetic component. Additionally, the rGC values of renal clearance of metformin, amoxicillin, and ampicillin were similar to previously reported rGC values for metabolism. By contrast, the rGC values of renal clearance for iohexol, digoxin, and terodiline were low (0.12-0.37). Renal clearance of these compounds occurs mainly through passive processes (e.g. glomerular filtration and passive secretion/reabsorption). The low rGC values of iohexol, digoxin and terodiline suggest that environmental factors may contribute to variation in their renal clearance.

The organic cation transporter, OCT1, is a major hepatic transporter that mediates the uptake of many organic cations from the blood into the liver where the compounds may be metabolized or secreted into the bile. Because OCT1 interacts with a variety of structurally diverse organic cations, including clinically used drugs as well as toxic substances (e.g., N -methylpyridinium, MPP + ), it is an important determinant of systemic exposure to many xenobiotics. To understand the genetic basis of extensive interindividual differences in xenobiotic disposition, we functionally characterized 15 protein-altering variants of the human liver organic cation transporter, OCT1, in Xenopus oocytes. All variants that reduced or eliminated function (OCT1-R61C, OCT1-P341L, OCT1-G220V, OCT1-G401S, and OCT1-G465R) altered evolutionarily conserved amino acid residues. In general, variants with decreased function had amino acid substitutions that resulted in more radical chemical changes (higher Grantham values) and were less evolutionarily favorable (lower blosum 62 values) than variants that maintained function. A variant with increased function (OCT1-S14F) changed an amino acid residue such that the human protein matched the consensus of the OCT1 mammalian orthologs. Our results indicate that changes at evolutionarily conserved positions of OCT1 are strong predictors of decreased function and suggest that a combination of evolutionary conservation and chemical change might be a stronger predictor of function.

The multidisciplinary UCSF Pharmacogenetics of Membrane Transporters project seeks to systematically identify sequence variants in transporters and to determine the functional significance of these variants through evaluation of relevant cellular and clinical phenotypes. The project is structured around four interacting cores: genomics, cellular phenotyping, clinical phenotyping, and bioinformatics. The bioinformatics core is responsible for collecting, storing, and analyzing the information obtained by the other cores and for presenting the results, in particular, for the genomic data. Most of this process is automated using locally developed software written in Python, an open source language well suited for rapid, modular development that meets requirements that are themselves constantly evolving. Here we present the details of transforming ABI trace file data into useful information for project investigators and a description of the types of data analysis and display that we have developed.

Background Twin studies have been used to determine the genetic contribution to variation in hepatic drug metabolism. In contrast, little is known about the contribution of genetic factors to variation in renal drug elimination. In this study, we used monozygotic twin pairs to assess the heritability of the clearance of the anti-diabetic agent, metformin, a drug that is eliminated exclusively in the kidneys. Methods We administered 850 mg metformin to six monozygotic twin pairs and collected plasma and urine samples over 24 hours. Concentrations of metformin were determined by LC-MS. A modified calculation was used for assessing the heritability (H) of renal clearance: H= (Vbetween pairs− Vwithin pairs)/Vbetween pairs, where V is variance. Results In this interim analysis, we observed that 95% of the variation in renal clearance of metformin could be attributed to genetic factors (H=0.95), with an overall correlation coefficient of 0.97. Conclusions While the formal heritability calculation will require the enrollment of dizygotic twin pairs, this initial finding indicates that genetic factors contribute substantially to the renal clearance of metformin. The degree of heritability in renal clearance is similar to that observed in classical twin studies of drugs that are eliminated by hepatic metabolism. Clinical Pharmacology & Therapeutics (2005) 77, P61–P61; doi: 10.1016/j.clpt.2004.12.123