The CEA/CD3-Bispecific Antibody MEDI-565 (MT111) Binds a Nonlinear Epitope in the Full-Length but Not a Short Splice Variant of CEA

MEDI-565 (also known as MT111) is a bispecific T-cell engager (BiTE®) antibody in development for the treatment of patients with cancers expressing carcinoembryonic antigen (CEA). MEDI-565 binds CEA on cancer cells and CD3 on T cells to induce T-cell mediated killing of cancer cells. To understand the molecular basis of human CEA recognition by MEDI-565 and how polymorphisms and spliced forms of CEA may affect MEDI-565 activity, we mapped the epitope of MEDI-565 on CEA using mutagenesis and homology modeling approaches. We found that MEDI-565 recognized a conformational epitope in the A2 domain comprised of amino acids 326–349 and 388–410, with critical residues F326, T328, N333, V388, G389, P390, E392, I408, and N410. Two non-synonymous single-nucleotide polymorphisms (SNPs) (rs10407503, rs7249230) were identified in the epitope region, but they are found at low homozygosity rates. Searching the National Center for Biotechnology Information GenBank® database, we further identified a single, previously uncharacterized mRNA splice variant of CEA that lacks a portion of the N-terminal domain, the A1 and B1 domains, and a large portion of the A2 domain. Real-time quantitative polymerase chain reaction analysis of multiple cancers showed widespread expression of full-length CEA in these tumors, with less frequent but concordant expression of the CEA splice variant. Because the epitope was largely absent from the CEA splice variant, MEDI-565 did not bind or mediate T-cell killing of cells solely expressing this form of CEA. In addition, the splice variant did not interfere with MEDI-565 binding or activity when co-expressed with full-length CEA. Thus MEDI-565 may broadly target CEA-positive tumors without regard for expression of the short splice variant of CEA. Together our data suggest that MEDI-565 activity will neither be impacted by SNPs nor by a splice variant of CEA

Statistical Models for the Analysis of Isobaric Tags Multiplexed Quantitative Proteomics

Mass spectrometry is being used to identify protein biomarkers that can facilitate development of drug treatment. Mass spectrometry-based labeling proteomic experiments result in complex proteomic data that is hierarchical in nature often with small sample size studies. The generalized linear model (GLM) is the most popular approach in proteomics to compare protein abundances between groups. However, GLM does not address all the complexities of proteomics data such as repeated measures and variance heterogeneity. Linear models for microarray data (LIMMA) and mixed models are two approaches that can address some of these data complexities to provide better statistical estimates. We compared these three statistical models (GLM, LIMMA, and mixed models) under two different normalization approaches (quantile normalization and median sweeping) to demonstrate when each approach is the best for tagged proteins. We evaluated these methods using a spiked-in data set of known protein abundances, a systemic lupus erythematosus (SLE) data set, and simulated data from multiplexed labeling experiments that use tandem mass tags (TMT). Data are available via ProteomeXchange with identifier PXD005486. We found median sweeping to be a preferred approach of data normalization, and with this normalization approach there was overlap with findings across all methods with GLM being a subset of mixed models. The conclusion is that the mixed model had the best type I error with median sweeping, whereas LIMMA had the better overall statistical properties regardless of normalization approaches

Role of the CEA splice variant in MEDI-565 mediated T cell activation and target cell killing.

<p>A, expression of full-length CEA and CEA splice variant proteins in CHO cells as determined by flow cytometry. Mouse IgG1, mouse IgG1 control antibody. B, CHO DHFR-, CHO FL, CHO SV and CHO FL+SV cells were tested for their susceptibility to be killed by CD3+ T cells from 3 individual donors in the presence of MEDI-565 at the indicated concentrations. EC₅₀ values listed indicate the mean value among 3 donors±standard error of the mean. p = 0.79 comparing cytotoxicity EC₅₀ values between CHO FL CEA and CHO FL+SV CEA cells. C, activation (increased cell surface CD25/IL-2R levels) of CD8+ T cells and D, activation of CD4+ T cells isolated from each of the 3 healthy donors was investigated concurrently with the cytotoxicity assays shown in panel B. p = 0.60 comparing CD8+ T cell activation EC₅₀ values between CHO FL CEA and CHO FL+SV CEA; p = 0.15 comparing CD4+ T cell activation EC₅₀ values between CHO FL CEA and CHO FL+SV CEA. MFI CD25-APC = mean fluorescence intensity of bound APC labeled, anti-human CD25 mAb. Experiment was repeated once with similar results.</p

Amino acid sequence alignment of the full-length CEA (Swiss-Prot P06731-1) and CEA splice variant proteins.

<p>Amino acids corresponding to the signal peptide, the six IgC-like domains, and the C-terminal peptide which is removed during the protein maturation process are over-lined with alternating solid or dashed lines. The epitope regions (aa 326–349 and 388–410) are enclosed by black boxes. The critical aa N³³³ involved in MEDI-565 binding is labeled with an arrow▴. Two polymorphisms in the epitope region are marked with a diamond ♦ (rs10407503; aa change Ala → Asp) and a circle • (rs7249230; aa change Glu → Lys).</p

Percent expression of full-length CEA and CEA splice variant transcripts in primary human tissues determined by qPCR.

<p>Percent expression of full-length CEA and CEA splice variant transcripts in primary human tissues determined by qPCR.</p

Critical amino acids in CEA involved in MEDI-565 binding.

<p>A, flow cytometry analysis of binding of MEDI-565 to CEA mutants targeting amino acids in segments A and C of the A2 domain. MEDI-565 did not bind to the variants in which the N³³³ of the A2 domain was mutated (KO_FTN and KO_NE), but bound well to the other mutants. B, a modeled structure of the A2 domain of CEA revealed two clusters of amino acids (V³⁸⁸G³⁸⁹P³⁹⁰E³⁹² and I⁴⁰⁸N⁴¹⁰) in segment C (green) that were spatially close to the critical aa N333 (red) in segment A (yellow), while segment B (cyan) was distal to aa N333. C, binding characteristics of MEDI-565 to CEA mutants with single or combinatorial mutations. Replacing N³³³ with Lys (KO_N) or Ala (N³³³ to A) disrupted the binding of MEDI-565 to the A2 domain. Mutating F³²⁶ or T³²⁸ to Ala reduced the binding of MEDI-565 (F³²⁶ to A, T³²⁸ to A). Knocking-out the amino acids V³⁸⁸G³⁸⁹P³⁹⁰E³⁹² and I⁴⁰⁸N⁴¹⁰ together from the A2 domain also reduced MEDI-565 binding (KO_VGPE+IN). Grafting of three amino acids F³²⁶T³²⁸N³³³ of segment A of the A2 domain in combination with six amino acids V³⁸⁸G³⁸⁹P³⁹⁰E³⁹² and I⁴⁰⁸N⁴¹⁰ of segment C of the A2 domain into the A3 domain (KI_FTN+VGPE+IN) resulted in MEDI-565 binding to a level comparable to that of the mutant encoding both segments A and C of the A2 domain together (KO_B/KI_A+C, KO_B encodes the same amino acids as KI_A+C).</p