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CARP: A High-Throughput Platform for Rapid VHH Discovery of Potent CAR-T Therapy

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Figure 1. CARP high throughput screening workflow: It begins hits discovery by using VHHMAb® platform, followed by AI-powered VHH library construction that encompasses antibodies with diverse epitopes and low immunogenicity. This is followed by pooling CAR-T clones, co-culturing with target cells, performing NGS of amplicons from the amplified CAR-T clones, and concluding with lead validation.

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Figure 2. The preparation of pooled CAR-T and serial proliferation by co-culturing with target+ cell lines. After CAR-T preparation (A), the percentage (B) and fold change (C) of CAR-T proliferation were assessed after co-culturing with H716 (CDH17 high), Colo205 (CDH17 medium), and HCT-116 (CDH17 negative) cells.

NGS²âÐò¼°ÔöÖ³ÄÜÁ¦ÅÅÐò£¬£¬£¬£¬£¬Í¨¹ý¶Ôɸѡ³ØÖÐÿÂÖ¹²×÷ÓýºóÓÅÓιú¼Ê¡¤UB8(ÖйúÓÎ)¼¯ÍŹٷ½ÍøվȺÌå¾ÙÐÐNGS²âÐò£¬£¬£¬£¬£¬»ñÈ¡¸÷¿Ë¡µÄÏà¶ÔƷòת±äÇúÏߣ¬£¬£¬£¬£¬²¢ÓÃбÂÊÄ£×Ó¾ÙÐÐÄâºÏ£¨Í¼3A¡¢3B£©£¬£¬£¬£¬£¬ÊµÏÖ¶ÔÊý°ÙºòÑ¡µÄ¿ìËÙ¶¨Á¿ÅÅÃû£»£»£»£»£»£»£»£»ËæºóʹÓò㼶¾ÛÀࣨͼ3C£©·Ö²ãɸѡ³ö¸ßDZÁ¦£¨¿ìËÙбÂÊÉÏÉý£©ÓëµÍDZÁ¦£¨ÇúÏßƽ»º»òϽµ£©VHHȺÌå¡£¡£¡£¡£¡£¡£¡£

Figure 3. NGS and slope analysis for VHH hit ranking. (A) Grouping information. (B) The proliferation curve shows the clone percentage of each round based on NGS, with the slope of the curve calculated through fitting. Three examples are shown to demonstrate different types of slopes. (C) Hierarchical clustering analysis for lead selection. (D) Twenty positive and six negative clones were selected for validation.

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Figure 4. Leads validation to confirm CARP accuracy in lead selection. Selected CART leads were co-culturing with H716 cells. (A) In the group of positive clones, 19 out of 20 demonstrated strong CAR-T expansion. (B) In the group of negative clones, 5 out of 6 showed no or minimal CART expansion. (C) For comparison, conventional CAR-T leads (selected based on good binding affinity to proteins and cells) were tested, and only 2 out of 5 exhibited strong CAR-T expansion.

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