Vectofusin-1 based T-cell transduction approach for developing murine CAR-T cells for cancer

Gene transfer into human and murine T-cells using viral-based approaches has several promising therapeutic applications including the production of chimeric antigen receptor T-cell (CAR-T) therapy. The generation of murine CAR-T is paramount to test and validate immunocompetent mouse models for CAR-T therapy. Several viral transduction enhancers already exist for gene therapy with few limitations. In this study, we tested vectofusin-1, a short cationic peptide, as a soluble transduction enhancer for gammaretroviral transduction for the generation of anti-CD19 murine CAR-T. We found that in comparison to Retronectin, Vectofusin-1 is an equally optimal transduction enhancer for the generation of murine CAR-T cells.


Introduction 42
The chimeric antigen receptor (CARs) are synthetic receptors that comprise a target 43 binding domain, derived mostly from a single-chain variable fragment of an antibody, a 44 hinge/transmembrane region, and a truncated CD3 zeta cytoplasmic domain with or without 45 costimulatory domains 1 . CAR T-cell therapy represents the latest advance in the field of 46 hematological malignancies with unprecedented response rates and survival outcomes 47 seen in patients with relapsed refractory lymphomas 2 . As such, there is a greater need to 48 produce CAR-T cells both for clinical use and for laboratory research purposes for in-vitro 49 and in-vivo validation studies. The generation of CAR-T cells through viral transduction can 50 be optimized through the addition of various cultures additives such as cationic polymers 51 (polybrene) 3 , dextran 4 , cationic lipids (lipofectamine 5 ) etc. Retronectin derived from 52 fibronectin, has recently been used for the generation of both human and murine CAR-T 53 cells for clinical applications 6,7 and research purposes. However, Retronectin based 54 transduction protocols are cumbersome as it needs to be surface coated before use and 55 therefore, a new soluble additive capable of enhancing infection is needed. Vectofusin-1, a 56 new cationic amphipathic peptide, is a soluble additive that has been used for transduction 57 of T-cells successfully; however, that has not been tested for the generation of murine 58 CAR-T cells. The goal of this study is to determine if a novel conjunction protein-peptide, 59 such as Vectofuscin-1, can achieve a similar transduction efficiency for the generation of 60 chimeric antigen receptor (CAR) T-cell in mouse T-cells. Here, we compare the vectofusin-61 1 based approach to a more traditional method of Retronectin protocols and provides step 62 by step approach to generating murine CAR-T cells through both approaches. 63 1. Activation beads can also be used for activation of T cells; however, they are costly 187 compared to coated antibody methods. We follow the manufacturer protocol with 188 beads to cell ratio at 1:1. Also, we must remove activation beads from T cells before 189 analysis or experimental use by pipetting T cells in a conical tube and thoroughly 190 pipetting up and down to remove the T-cells sticking to beads. Expose the tube to a 191 magnet for 2 min and unbound T cells can be decanted from the tube exposed to the 192 magnet. For our experiments, we obtained stable virus producing Phoenix-Eco cell lines from 197

Reagents
Dr. Marco L Davila's lab to generate gamma retrovirus (generous gift). 198 3. Human IL-2 can be used for both mouse and human T-cells. 199

Statistical Analysis: 200
Significance of variation between groups was evaluated using a non-parametric two-tailed 201 Student's t test. Test for differences between proportions was performed using two sided 202 Fisher's exact test with p ≤ 0.05 considered significant. 203

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Impact of activation beads versus plated antibody on transduction efficiency 207 Firstly, we wanted to evaluate the impact of beads versus plated antibodies on the 208 activation of murine T-cells and subsequent transduction efficiency. We took mice spleens 209 (n=3) and murine T-cells were isolated as detailed in the protocol above. 5x10 6 T-cells were 210 each individually activated with either the mouse T-cell activator beads or plated anti-211 Cd3/CD28 antibodies. Subsequently, we performed Retronectin based transduction assays 212 with same amount of harvested viral supernatants (3ml) in each well at 24 hours and 48 213 hours. Viral supernatant was obtained from the stable virus producer cell lines and were 214 pooled before transduction step. This allowed us to make sure that the viral titres were 215 similar between each individual experiments and step. 216 Our CAR plasmid vector is based on SFG plasmid backbone with anti-mouse CD19 CAR 217 sequence followed by a T2a and GFP sequence as shown in Figure 1A. We evaluated the 218 transduction efficiency in murine T cells by expression of Green fluorescent protein (GFP) 219 by flow cytometry on FITC channel. We found that by Retronectin based transduction 220 assays, similar transduction efficiency was generated between beads (mean=60.40 %, n= 221 3) and plated antibody anti-CD3/CD28 (mean=55.4%, n=3) T-cell activation methods as 222 shown in the figure 1B and 1C (p=0.14, unpaired t-tests, two-tailed).

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Retronectin is a better transduction enhancer compared to Vectofusin-1 for murine CAR-T 225 Next, we compared the Retronectin versus Vectofusin-1 as a transduction enhancer for 226 generation of murine CAR-T cells. For direct comparison, we activated murine T cells 227 isolated from mouse spleens (n=3) with plated anti-CD3/CD28 antibodies. Subsequently, 228 we performed viral transduction on 5X10 6 T-cells each on day 1 and day 2 with either 229 retronectin or vectofusin-1. We found that vectofusin-1 generated lower transduction 230 efficiency (mean 43.33, n=3) compared to Retronectin (mean=55.57, n=3, p-value 0.001, 231 paired two tailed t-tests) as shown in Figure 2. 232 233

CAR-T cells proliferation and expansion is optimal with Vectofusin-1 234
Next, we also evaluated the absolute number of cells at day 5 after transduction with the 235 help of Countess FL automated Cell Counters and computed the median fold expansion 236 from the baseline. We found that by the end of day 5, fold expansion of total T-cells in the 237 media were significantly higher with vectofusin-1 (1.9333 ±0.141 fold) compared to 238 Retronectin (1.4667 ±0.0706) transduction (p=0.008).
In our study, we compared vectofusin-1 and Retronectin as transduction enhancers for the 242 generation of murine anti-CD19 CAR-T. We found that the vectofusin-1 generated lesser 243 CAR-T transduction efficiency, as assessed by GFP expression, compared to Retronectin. 244 However, the absolute number of CAR-T cells generated was approximately equal between 245 the two groups due to higher expansion and proliferation of total T-cells under vectofusin-1. 246 The reason for the increased total number of T-cells generated with vectofusin-1 is unclear 247 to us. It is possible that Retronectin labeled plates adhere to T-cells more tightly after spin-248 down post-transduction and probably limit their mobility and proliferation to a small extent. 249 In conclusion, vectofusin-1 leads to optimal murine CAR-T production, similar to 250 Retronectin for performing in-vitro and in-vivo mouse validation studies.