Bispecific monoclonal antibodies (bsabs) recognizing both CD3 and a tumor antigen can redirect T-cell-mediated cytotoxicity toward cells bearing that antigen. Such bsabs have been shown to be more effective than monospecific monoclonal antibodies (MoAbs) at preventing tumor growth in animal models of B-cell malignancy. The current studies describe the production and preliminary evaluation of a bsab designed to induce the lysis of malignant human B cells by human T cells. The bsab was obtained from a hybrid-hybridoma cell line produced by fusing OKT3-secreting hybridoma cells with hybridoma cells that secrete 1D10. 1D10 is an MoAb that recognizes an antigen found on a majority of malignant human B cells that has not been detected to a significant degree on normal resting or activated lymphocytes. High performance liquid chromatography (HPLC) was used to separate bsab from monospecific antibodies that were also present in the hybrid-hybridoma antibody product. The bsab was then evaluated in vitro for its ability to induce lysis of malignant B cells by activated T cells. The bsab consistently induced extensive lysis in vitro of 1D10 (+) cells, including both cell lines and cells obtained from patients with a variety of B-cell malignancies. No such effect was seen with activated T cells alone or activated T cells with monospecific antibody. No increased lysis was seen with 1D10 (-) cell lines. The bsab also mediated lysis of malignant B cells by autologous T cells. We conclude bsab containing an OKT3 arm and a 1D10 arm can induce T-cell-mediated lysis in a manner that is both potent and specific. This supports further evaluation of this bsab as a potential immunotherapy of B-cell malignancy.

Introduction: Pathologic complete response (pCR) to neoadjuvant chemotherapy (NAC) is associated with improved long-term outcomes. Vitamin D (VD) deficiency has been associated with carcinogenesis as well as poorer outcomes in breast cancer (bc) particularly in luminal-type bc. In addition, replacement of VD during adjuvant chemotherapy was found to improve disease-free survival. However, VD deficiency was not associated with pCR following NAC in one series which included patients (pts) with human epidermal growth factor receptor-2 (HER2) negative bc only. We report the relationship between VD deficiency and pCR in a cohort that includes all bc subtypes and report how VD deficiency varied by clinical and pathologic parameters. Methods: Patients (pts) prospectively enrolled in the University of Iowa Breast Molecular Epidemiologic Resource between 2010-14 with invasive bc, receiving at least one cycle of NAC, and who had serum collected at enrollment or during NAC were eligible. VD deficiency was defined as < 20 ng/dl. pCR was defined as no residual invasive disease in breast and lymph nodes. Patients were stratified by their BMI as normal (≤ 25 kg/m2) or overweight-obese (>25 kg/m2). To investigate the relationship between VD, clinical parameters, and NAC outcomes, chi-square, Fisher's exact, and t-tests were used. Results: 73 pts were eligible. 52 (71%) pts had Stage I-II bc. All pts received a combination of taxanes with other agents (Cytoxan, 5FU, Carboplatin, Gemcitabine) with or without an anthracycline or anti-HER 2 therapy. 62/73 (85%) received an anthracycline. 65/73 (89%) received at least 75% of intended NAC. pCR was achieved in 25/73 (34%) pts. VD deficiency was found in 17/73 (23%) pts and was more frequent among overweight-obese pts (Table 1). VD deficiency occurred significantly more often in estrogen receptor (ER)+/progesterone receptor (PR)+/HER2- and ER+ or PR+ bc but not in HER2+ bc. Results suggest VD deficient pts may be at increased odds of not achieving a pCR (OR=3.02, p=0.10). In this cohort, VD deficient pts were at increased odds of having residual disease in the breast (OR=3.76, p=0.05), but not in the nodes (OR=1.43, p=0.53), (Table 2). Conclusion: Being overweight-obese was associated with VD deficiency and may warrant screening in this group. VD deficiency was significantly associated with ER+/PR+/HER2-bc and ER+ or PR+ disease. Notably, these pts are candidates for bone-injuring anti-estrogen agents as part of their overall course of therapy. VD deficiency was not significantly associated with pCR after NAC though a trend was suggested. Table 1: VD and clinical parameters   VD Sufficient VD Deficientp  N=56N=17 Age (mean in years) 52470.16BMI≤2526(46%)3(18%)0.03 >2530(54%)14(82%) Ellis-Ellison grade1-224(43%)7(41%)0.90 332(57%)10(59%) Tumor receptorER+/PR+/HER2-15(27%)10(59%)0.02 ER+ or PR+/HER2+6(11%)3(18%)0.43 ER-/PR-/HER2+10(18%)0(0%)0.10 ER-/PR-/HER2-17(31%)3(18%)0.36 ER+ or PR+29(52%)14(82%)0.02 HER2+16(84%)3(16%)0.53 Table 2: VD and NAC Outcomes   VD SufficientVD Deficientp  N=56N=17 pCRNo34(61%)14(82%)0.10 Yes22(39%)3(18%) Breast Residual DiseaseNo25(45%)3(18%)0.05 Yes31(55%)14(84%) Node Residual DiseaseNo28(50%)7(41%)0.52 Yes28(50%)10(59%)  Citation Format: Raman R, Link BK, Mott SL, Schroeder MC, Thomas A. Vitamin D deficiency in various breast cancer subtypes and its impact on response to neoadjuvant chemotherapy in operable breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-08-48.