Preclinical Evaluation of Anti-ROR1 CAR T-cells Employing a ROR1 … SITC 2020 ROR1 CAR... ·...
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Authors: Charles E. Prussak 1 , Christopher Oh 1 , Juliana Velez Lujan 1 , Sharon Lam 1 , Jieyu Zhang 1 , Jayde Fernandez 1 , Navyaa Sinha 1 , Jessica Pham 1 , Haoming Shi 1 , Caitlin Shin 1 , Gunnar F. Kaufmann 2 , George F. Widhopf II 1 , Ezra E.W. Cohen 1 , Thomas J. Kipps 1 ; 1) University of California, San Diego Moores Cancer Center, La Jolla, CA; 2) Oncternal Therapeutics, Inc., San Diego, CA Conclusions : • Single dose of ROR1 CAR T-cells expanded in MEC1-ROR1 xenografts and homed to disease sites. • By week four, rapidly-expanding leukemia cells were cleared from major tissue reservoirs, including bone marrow, kidneys and spleen. • CAR T-cell-treated animals survived longer than 90 days vs 21 days for animals in control groups. • CAR -T-cells were highly active and detected in mouse tissues more than 2 months after injection. These studies are supported by a grant from the California Institute of Regenerative Medicine (CIRM) TRAN 10258 Future Directions for Research: Results Figure 4. Bioluminescence imaging of MEC1-ROR1 cells following treatment with ROR1 CAR T-cells. Mice treated with 3e6 CAR T reduced the leukemic burden to background levels by day 30 and controlled disease for remainder of study. Animals in the control groups (untreated, ATC or lower 1e6 dose) had to be sacrificed on day 20. Figure 5. Detection of CAR T-cells in mouse tissues. Following CAR T administration, animals were sacrificed on days 11 (Panel A), and 25 (Panel B), blood and organs collected and subjected to flow analysis for CAR expression and confirmatory ROR1 binding activity. The CAR T-cell number was substantially greater in mice bearing MEC-1 ROR1 cells (CAR + MEC1-ROR1) vs control (CAR only) demonstrating elevated expansion of CAR T-cells in animals with tumor burden. Bars represent the mean values from the five mice in each group and error bars represent the S.E of the mean. Introduction We generated chimeric antigen receptor T (CAR T) cells that target the extracellular domain of ROR1, which is expressed on many human cancers but not on post-partum tissues. The anti-ROR1 CAR utilizes the humanized binding domain of UC-961 (cirmtuzumab), which currently is being evaluated in Phase 1/2 studies for patients with hematologic and solid-tumor malignancies (NCT 02222688, 02776917). Cirmtuzumab has demonstrated high affinity for human ROR1 with no off-target binding 1 , a prolonged half-life, and excellent safety profile in Phase 1 clinical studies 2 . The humanized scFv of cirmtuzumab represents an excellent ROR1 targeting moiety for use in CAR-based cell therapies. We developed multiple candidate 2 nd and 3 rd generation CAR constructs with different signaling and spacer regions. To evaluate these constructs, we generated candidate CAR T-cells in a serum- free growth media supplemented with IL-2. Using this protocol, we produced CAR T-cells from over 20 healthy donors and examined their activity against lymphoid cancers in vitro using chromium release and impedance assays. The 2 nd generation CARs using the 4-1BB co- stimulation domain demonstrated activity even at low effector to target (E:T) ratios, eliminating both primary B-cell leukemias and lymphomas, as well as tumor cell lines that express the ROR1 target. Methods To evaluate ROR1 CAR T-cells, we created a luciferase/RFP lentiviral expression vector to generate labeled human MEC1-ROR1 leukemia cells 3 . When injected into immune-deficient NSG mice, MEC1-ROR1 cells infiltrate the marrow (femur), kidney, spleen, and liver, resulting in progressive and fatal leukemia/lymphoma. From multiple 2 nd generation CAR T-cell products, we selected the CAR with 4-1BB and CD3z signaling domains that demonstrated the best activity in in vitro assays. To introduce the CAR into cells, we created lentiviral vectors encoding the ROR1 CAR (Figure 1). Results of these studies strongly encourage clinical studies evaluating safety and efficacy of our ROR1-directed CAR T cell therapy for patients with hematological malignancies. Figure 1. Structure of the anti-human anti-ROR1 CAR. The 2 nd generation CAR employing the humanized anti-ROR1 cirmtuzumab variable regions of heavy chain (VH) and light chain (VL). Figure 2. In vitro cell killing activity of ROR1 CAR T-cells in a 4h chromium release assay (left) and 120h Acea impedance assay (right). ROR1 CAR T-cell products were generated from 2 healthy donors as described above. The generated CAR T-cells were tested at the indicated E:T ratios. The anti-ROR1 CAR T-cells demonstrated high and specific cytotoxicity without significant killing of ROR1-negative target cells [less than 10% of specific killing (data not shown)]. ATC = activated T-cells The in vitro killing activity of this highly active and specific CAR product is shown below (Figure 2). References: 1) Choi, Clin Lymphoma Myeloma Leuk, 2015; 2) Choi, Cell Stem Cell, 2018; 3) Yu et al., J Clin Invest, 2015 Figure 3. Bioluminescence imaging of mice inoculated with MEC1-ROR1 cells and with ROR1 CAR T-cells. Animals treated with CAR T-cells had reduced disease burden compared to controls. The highest dose (3e6 CAR Ts) cohort had only minimal amounts of disease at the end of the study. Preclinical Evaluation of Anti-ROR1 CAR T-cells Employing a ROR1 Binding scFv Derived From the Clinical Stage mAb Cirmtuzumab CAR + MEC1-ROR1 CAR Only 0 20 40 60 80 100 % CD3+ CAR + MEC1-ROR1 CAR Only 0 20 40 60 80 100 % CD3+ Blood Spleen Liver Kidney Bone Marrow CAR + MEC1-ROR1 CAR Only 0 20 40 60 80 100 % ROR1 Ig Binding CAR + MEC1-ROR1 CAR Only 0 20 40 60 80 100 % ROR1 Ig Binding A B Days ET0.33 ET1 ET3 0 20 40 60 80 100 % Specific Lysis Unmet Need: Emerging CAR T Issues Treatment failures • Increasing number of patient relapses following CAR T therapy, frequently due to mutations or loss of target tumor antigen (e.g. CD19), evading CAR T-cells Safety concerns • Persistent CAR T safety issues including deaths potentially related to activation by normal cells expressing the target antigen (“on-target/off-tumor”) Advantages to Targeting ROR1 Potential for fewer antigen negative relapses • ROR1 expression associated with aggressive tumor phenotype • ROR1 mutation or antigen loss might render cancer cells less aggressive and susceptible to chemotherapy Potential safety advantages • Cirmtuzumab did not bind to normal human tissues in GLP tissue cross-reactivity studies • No serious adverse events related to cirmtuzumab-only reported in clinical studies Rationale: CAR T Targeting ROR1 Designed to Avoid Common CAR T Challenges Activated T-cells CAR T donor 1 CAR T donor 2 No addition Activated T-cells CAR T donor 1 CAR T donor 2 Target cells: MCF-7-ROR1 Target cells: MEC1-ROR1 cells
Transcript of Preclinical Evaluation of Anti-ROR1 CAR T-cells Employing a ROR1 … SITC 2020 ROR1 CAR... ·...
Authors: Charles E. Prussak1, Christopher Oh1, Juliana Velez Lujan1, Sharon Lam1, Jieyu Zhang1, Jayde Fernandez1, Navyaa Sinha1, Jessica Pham1, Haoming Shi1, Caitlin Shin1, Gunnar F. Kaufmann2, George F. Widhopf II1, Ezra E.W. Cohen1, Thomas J. Kipps1; 1) University of California, San Diego Moores Cancer Center, La Jolla, CA; 2) Oncternal Therapeutics, Inc., San Diego, CA
Conclusions:• Single dose of ROR1 CAR T-cells expanded in
MEC1-ROR1 xenografts and homed to disease sites.
• By week four, rapidly-expanding leukemia cellswere cleared from major tissue reservoirs, includingbone marrow, kidneys and spleen.
• CAR T-cell-treated animals survived longer than 90days vs 21 days for animals in control groups.
• CAR -T-cells were highly active and detected inmouse tissues more than 2 months after injection.
These studies are supported by a grant from the California Institute of Regenerative Medicine (CIRM) TRAN 10258 Future Directions for Research:
Results
Figure 4. Bioluminescence imaging of MEC1-ROR1cells following treatment with ROR1 CAR T-cells.Mice treated with 3e6 CAR T reduced the leukemicburden to background levels by day 30 andcontrolled disease for remainder of study. Animalsin the control groups (untreated, ATC or lower 1e6dose) had to be sacrificed on day 20.
Figure 5. Detection of CAR T-cells in mousetissues. Following CAR T administration,animals were sacrificed on days 11 (Panel A),and 25 (Panel B), blood and organs collectedand subjected to flow analysis for CARexpression and confirmatory ROR1 bindingactivity. The CAR T-cell number wassubstantially greater in mice bearing MEC-1ROR1 cells (CAR + MEC1-ROR1) vs control (CARonly) demonstrating elevated expansion of CART-cells in animals with tumor burden.Bars represent the mean values from the five micein each group and error bars represent the S.E of themean.
Introduction We generated chimeric antigen receptor T (CAR T) cells that target the extracellulardomain of ROR1, which is expressed on many human cancers but not on post-partum tissues.The anti-ROR1 CAR utilizes the humanized binding domain of UC-961 (cirmtuzumab), whichcurrently is being evaluated in Phase 1/2 studies for patients with hematologic and solid-tumormalignancies (NCT 02222688, 02776917). Cirmtuzumab has demonstrated high affinity forhuman ROR1 with no off-target binding1, a prolonged half-life, and excellent safety profile inPhase 1 clinical studies2. The humanized scFv of cirmtuzumab represents an excellent ROR1targeting moiety for use in CAR-based cell therapies.
We developed multiple candidate 2nd and 3rd generation CAR constructs with different signalingand spacer regions. To evaluate these constructs, we generated candidate CAR T-cells in a serum-free growth media supplemented with IL-2. Using this protocol, we produced CAR T-cells fromover 20 healthy donors and examined their activity against lymphoid cancers in vitro usingchromium release and impedance assays. The 2nd generation CARs using the 4-1BB co-stimulation domain demonstrated activity even at low effector to target (E:T) ratios, eliminatingboth primary B-cell leukemias and lymphomas, as well as tumor cell lines that express the ROR1target.
Methods To evaluate ROR1 CAR T-cells, we created a luciferase/RFP lentiviral expression vectorto generate labeled human MEC1-ROR1 leukemia cells3. When injected into immune-deficientNSG mice, MEC1-ROR1 cells infiltrate the marrow (femur), kidney, spleen, and liver, resulting inprogressive and fatal leukemia/lymphoma. From multiple 2nd generation CAR T-cell products, weselected the CAR with 4-1BB and CD3z signaling domains that demonstrated the best activity inin vitro assays.To introduce the CAR into cells, we created lentiviral vectors encoding the ROR1 CAR (Figure 1).
Results of these studies strongly encourage clinical studies evaluating safety and efficacy of our ROR1-directed CAR T cell therapy for patients with hematological malignancies.
Figure 1. Structure of the anti-human anti-ROR1 CAR. The 2nd generation CAR employing the humanized anti-ROR1cirmtuzumab variable regions of heavy chain (VH) and light chain (VL).
Figure 2. In vitro cell killing activity of ROR1 CAR T-cells in a 4h chromium release assay (left) and 120h Aceaimpedance assay (right). ROR1 CAR T-cell products were generated from 2 healthy donors as described above. Thegenerated CAR T-cells were tested at the indicated E:T ratios. The anti-ROR1 CAR T-cells demonstrated high andspecific cytotoxicity without significant killing of ROR1-negative target cells [less than 10% of specific killing (data notshown)]. ATC = activated T-cells
The in vitro killing activity of this highly active and specific CAR product is shown below (Figure 2).
References: 1) Choi, Clin Lymphoma Myeloma Leuk, 2015; 2) Choi, Cell Stem Cell, 2018; 3) Yu et al., J Clin Invest, 2015
Figure 3. Bioluminescence imaging of mice inoculated with MEC1-ROR1 cells and with ROR1CAR T-cells. Animals treated with CAR T-cells had reduced disease burden compared tocontrols. The highest dose (3e6 CAR Ts) cohort had only minimal amounts of disease at the endof the study.
Preclinical Evaluation of Anti-ROR1 CAR T-cells Employing a ROR1 Binding scFv Derived From the Clinical Stage mAb Cirmtuzumab
CAR + MEC1-ROR1 CAR Only0
20
40
60
80
100
% C
D3+
CAR + MEC1-ROR1 CAR Only0
20
40
60
80
100
% C
D3+
Blood
SpleenLiverKidneyBone Marrow
CAR + MEC1-ROR1 CAR Only0
20
40
60
80
100
% R
OR1
Ig B
indi
ng
CAR + MEC1-ROR1 CAR Only0
20
40
60
80
100
% R
OR1
Ig B
indi
ng
A
B
Days
ET0.33 ET1 ET30
20
40
60
80
100
% S
peci
fic L
ysis
Unmet Need: Emerging CAR T IssuesTreatment failures• Increasing number of patient relapses
following CAR T therapy, frequently due to mutations or loss of target tumor antigen (e.g. CD19), evading CAR T-cells
Safety concerns• Persistent CAR T safety issues including
deaths potentially related to activation by normal cells expressing the target antigen (“on-target/off-tumor”)
Advantages to Targeting ROR1Potential for fewer antigen negative relapses• ROR1 expression associated with aggressive
tumor phenotype• ROR1 mutation or antigen loss might render
cancer cells less aggressive and susceptible to chemotherapy
Potential safety advantages• Cirmtuzumab did not bind to normal human
tissues in GLP tissue cross-reactivity studies• No serious adverse events related to
cirmtuzumab-only reported in clinical studies
Rationale: CAR T Targeting ROR1 Designed to Avoid Common CAR T Challenges
Activated T-cellsCAR T donor 1CAR T donor 2
No additionActivated T-cellsCAR T donor 1CAR T donor 2
Target cells: MCF-7-ROR1Target cells: MEC1-ROR1 cells
