Developing an Arrayed CRISPR-Cas9 Co-Culture Screen for Immuno-Oncology Target ID.

Immunotherapies including PD-L1 blockade have shown remarkable increases in the T cell-directed antitumor response; however, efficacy is seen only in a minority of patients. Recently, pooled CRISPR-Cas9 knockout (CRISPRn) screens in tumor/immune co-culture systems have identified a number of genes that confer resistance to T cell killing in pathways including antigen presentation and cytokine signaling, providing insight into tumor mechanisms that cause resistance to immunotherapies. The development of an arrayed CRISPRn screen in a tumor/immune co-culture system would allow the identification of novel targets for immuno-oncology, characterization of hits from pooled screens, and multiple assay endpoints to be measured per gene. Here, a small-scale arrayed CRISPRn screen was successfully developed to investigate the effects on a co-culture of T cells and Cas9-expressing PC9 lung adenocarcinoma cells modified to express anti-CD3 antibody on the cell surface (PC9-OKT3 T cell system). A focused CRISPRn library was designed to target genes involved in known resistance mechanisms (including antigen presentation, cytokine signaling, and apoptosis) as well as genes involved in immune synapse interactions. The viability of PC9 cells was assessed in two-dimensional adherent co-cultures via longitudinal imaging analysis. Knockout of epidermal growth factor receptor (EGFR) and PLK1 in tumor cells cultured alone or with T cells resulted in increased tumor cell death, as expected, whereas knockout of the test gene ICAM1 showed subtle donor-specific resistance to T cell killing. Taken together, these data provide proof of concept for arrayed CRISPRn screens in tumor/immune co-culture systems and warrant further investigation of in vitro co-culture models.

Removal of therapeutic anti-lymphocyte antibodies from human sera prior to anti-human leukocyte antibody testing.

Both monoclonal (e.g. Orthoclone (OKT3), rituximab) and polyclonal (e.g. ATGAM, Thymoglobulin (Thymo)) anti-lymphocyte Abs (ALAs) are used extensively in organ transplantation for immunosuppression induction, desensitization, and treatment of acute rejection. ALAs often interfere with post transplant immunologic monitoring. We describe a method that uses magnetic beads to selectively remove ALAs from patient serum. Rabbit anti-mouse Fc-specific (180 mug), or rabbit anti-mouse Fab-specific (180 microg), or rabbit anti-horse heavy and light chain-specific and rabbit anti-horse F(ab')2 (200 microg) (Jackson Immunoresearch) was adsorbed to 6.7 x 10(8) Dynabeads M-280 conjugated with sheep anti-rabbit IgG (Dynal Biotech). Fifty microliters of normal human serum (NHS) with 2 microg/ml of OKT3 or 100 microg/ml ATGAM, Thymo, or rituximab were incubated with conjugated beads for several incubations. NHS containing ALAs before and after treatment by the protocol were incubated with human lymphocytes and labeled with FITC-antibody to immunoglobulin of the species used to produce the particular ALA. Residual ALA was determined using flow cytometry. Average median channel for serum with or without ALA was 11.1 and 0.120, respectively for OKT3; 64.4 and 0.344 for ATGAM; 108.5 and 0.200 for Thymo; and 1022.5 and 11.4 for rituximab. Treatment lowered the median channel for serum with OKT3 to 0.103, 0.309 for ATGAM, 0.199 for Thymo, and 12.1 for rituximab. ALAs can be effectively removed from serum by the use of magnetic beads conjugated with Ab specific for ALA thereby permitting immunologic monitoring without interference.

In vitro characterization of five humanized OKT3 effector function variant antibodies.

Orthoclone OKT 3 (mOKT3) is a highly effective agent for the reversal of steroid-resistant renal allograft rejection. However, its wider use has been limited by the development of a human anti-mouse antibody response (HAMA) and by the "cytokine release syndrome" (CRS). CRS has been associated with T cell/monocyte activation and, secondarily, with activation of the complement cascade. These processes are mediated through Abs' Fc regions by their abilities to cross-link T cells and mononuclear cells and to activate complements. To alleviate these problems, a group of five huIgG1- and huIgG4-based OKT3 wild-type antibodies and their corresponding Fc mutants with altered residues at amino acids 234, 235, and 318, reported to be required for FcgammaRI and FcgammaRII binding and complement fixation, were constructed. Characterization of these humanized OKT3 Abs, denoted huOKT3gamma1, huOKT3gamma4, huOKT3gamma1(A(234), A(235)), huOKT3gamma4(A(234), A(235)), and huOKT3gamma1(A(318)), has demonstrated that huOKT3gamma1(A(234), A(235)) and huOKT3gamma4(A(234), A(235)), and have at least a 100-fold reduced binding to FcgammaRI and FcgammaRII. As expected, they are much less potent in the induction of T cell activation and cytokine release, yet retain in vitro immunosuppressive effects as potent as those of mOKT3. Unexpectedly, while huOKT3gamma1(A(318)) did not show any reduction in its ability to bind C1q and to fix a complement, huOKT3gamma1(A(234), A(235)) was completely inactive. The in vitro characteristics of huOKT3gamma1(A(234), A(235)) are consistent with recent in vivo studies, in which this Ab showed greatly reduced HAMA and CRS with the retention of its ability to reverse ongoing graft rejection in man.

Ion exchange resins for the purification of monoclonal antibodies from animal cell culture.

We have compared various ion exchangers for monoclonal antibody (MAb) purification using different starting materials such as ascitic fluid and cell culture supernatant. Twelve cation and anion exchange resins were tested so far. Purification of MAbs with regard to the starting material is described. In well-defined conditions of adsorption (20 mM MES buffer, pH 6.50), one purification step based on cation-exchange chromatography is generally sufficient to achieve at least 90% purity of the MAb, even when produced by animal cell culture. Cation-exchange supports exhibit higher capacity for MAbs compared to anion exchangers. Among the cation exchangers tested, we have selected the cross-linked matrix S Sepharose FF for its large specificity and capacity for MAbs. Considering these key parameters and also the good mechanical resistance of the S Sepharose FF, we describe how, by varying the flow rate, sample concentration, and size of the column, the productivity may be improved in a monoclonal antibody purification process. Finally, a general 'gram scale' purification protocol of MAbs produced by animal cell cultures is proposed. This protocol, based on economical adsorption conditions and three steps of elution (100 mM, 200 mM and 1 M NaCl), allows the recovery of highly purified MAbs.