Peer review of: Engineering AvidCARs for combinatorial antigen recognition and reversible control of CAR (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
Salzer et al. present a new spin on the CAR platform that is based on coupling reduced-affinity 
targeting domains to exogenous CAR dimerization. The authors propose this as a better system 
than other published modalities for combinatorial targeting. 
 
Overall, the engineering work is nicely done and well thought-out. However: 
 
Major comments: 
1. The concept needs to be fleshed out with more examples of targeting domains. I do not think it 
is sufficient to use only the monovalent E11.4.1 binder. Also, more models with scFv to show how 
generalizable this concept is. 
2. One of the major claims here is that the AVID-CAR system can discriminate single- from double-
antigen-expressing cells. This is done quite well in vitro but the in vivo work is not convincing. The 
authors would need to show in the same animal that single antigen tumor cells are ignored while 
double antigen cells are killed. The claim on page 12 line 282-283 is not actually supported by 
their data. 
3. Page 5, lines 112-124. This claim would be greatly enhanced by actual experimental data to test 
their mathematical prediction. 
4. Page 6, line 136. Since the data suggest that the low affinity AvidCAR confers high sensitivity 
only in settings where the ligands are present in dimeric form, this makes me question the 
relevance of this platform to real life. What two different tumor antigens are present in dimeric 
form? 
5. Page 11 lines 262-264 and Fig 5d,e. Here the results are quite marginal and the follow-up is 
short. Therefore the data are not particularly convincing. 
6. Page 12, line 301-302. There actually are examples of bispecific CARs that show “AND” gating 
logic. For example, Kloss et al, Nat Biotech 2013. 
7. Page 13, line 320-322. Increasing tumor specificity by requiring a dependence on two different 
antigens (i.e. AND gating) opens not one but two doors for tumor escape through antigen loss (or 
downmodulation). Therefore I am not sure that this design is ultimately advisable. 
 
Minor comments: 
1. It would be interesting to see more in-depth mechanistic studies. For example, page 8 line 185, 
are there differences in the activation of signaling proteins that might explain why the different 
designs have disparate effects on killing vs cytokine production? 
2. Fig 2d. although there is no statistical significance in the high and intermediate affinity 
comparisons, I wonder whether with additional repeats of this experiment, these results will hold 
up. There is a clear trend to reduced activity in the ser-BBz constructs. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
The one of major challenges of CART therapy is controlling the toxicities associated with the 
therapy due the lack of cancer specific targets and treatment associated side effects, such as 
tumor lysis syndrome and cytokine storm, as well as off target effects. Developing novel ON-switch 
and AND-gate systems that can not only effectively control specificity of CARs, but also minimally 
affect efficacy of the therapy is critically important to advance the field of CART therapy. 
In this report, the authors developed a novel avidity-controlled CAR (AvidCAR) platform with 
inducible and logic control functions that enable to control CART function via ON-switch and AND-
gate mechanisms. 
 
However, the data present in current version of manuscript are far from being convincing and solid 

enough to show that the systems are optimized to be potentially useful. Major changes have to be 
made before publication. 
 
Some suggestion for revision: 
 
1. Based on the data shown in Figure 2F and Supp. 4B, that suggest: a. Overall, AvidCAR activities 
are lower than high-affinity CAR; b. At lower antigen density, AvidCAR still reactive. C. At high 
antigen density, there is nearly no differences between AvidCAR and monomeric low affinity Ser-
CAR for both lytic and cytokine production. Therefore, it seems that monomeric low affinity Ser-
CAR is safer and more specific to high antigen density (equivalent to tumors), where spares low 
antigen density (equivalent to normal tissues). 
 
2. The tumor lines that express EGFR or Her2/neu at different levels should be tested to 
demonstrate that AvidCAR can differentially recognize tumors that express antigen at high levels. 
 
3. The authors conclude that AvidCAR cannot use scFv, but only in FV form, which might be 
misleading, since only one scFv (4D5-5) was tested. 
 
 
4. AvidCAR works with only 4-1bb domain based CAR, which also imply that the systems can be 
affected by some unpredicted and uncontrollable factors and makes this platform less reliable and 
useful. 
 
5. Only Her2/neu high expressing cell lines were used in Affibody mutant R10A AvidCAR work. 
Tumors, or cell lines that express Her2/neu at different levels should be tested, to exclude the 
possibility that the dimerized R10A AvidCAR has no discrimination for antigen expressions. 
 
6. Although cytokine productions were used in some experiments, the in vitro test was mainly 
relayed on CTL (killing) assay. All the CTL assays were used fixed single E:T ratio, which cannot 
exactly tell the difference of the killing ability of T cells, especially at high E:T ratio, such as for 
Figure 5A&B, E:T ratio of 10:1 was used. The lytic activity at different E:T ratio should be shown 
for most of the experiments. 
 
 
7. The Nalm6 model used in this report was much weaker than most of the reports by other people 
who used 1e6 Nalm6 (i.v.) and treatment started at day 5 or 7 later with 1 to 10 million of 
transduced CART cells. While in this report, 5e5 Nalm6 with treatment started at 3 days (Figure 
5C) or 5 days (Figure 6C) with 10 million of CART cells, with such condition, 10 million FMC63 
CD19 CART cells are far more than enough to completely cure the mice, while AvidCAR only 
showed minimum effect on controlling tumor growth. That’s might be the reasons why the 
experiments (for both Figure 5C and 6C) were terminated at extremely early after treatments. 
 
 
8. For Nalm6 model, long time (usually two to three month) follow-up and survival cures are 
required. 
 
 
9. Since the systems are mainly developed for the treatment of solid tumors and the targets 
developed in this report are EGFR and Her2/neu, the system should be tested in solid tumor 
models.